Primary cilia are fascinating, evolutionary conserved cell organelles involved in sensing of extracellular signals during development and adulthood and are consequently considered to function as the “antenna of the cell.” Morphologically, primary cilia also remind of an antenna and form a protrusion that sticks out of the cell. This protrusion consists of a nine-duplet microtubular filament ring that arises from the mother centriole and is covered by the ciliary membrane. The ciliary membrane contains various receptor proteins and consequently a growing number of signaling pathways originate at primary cilia. These comprise Hedgehog (HH) signaling, transforming growth factor (TGF)-β signaling, G protein-coupled receptor (GPCR) signaling, platelet-derived growth factor receptor (PDGFR)-α, mechanistic target of rapamycin (mTOR) signaling, Hippo signaling, Notch signaling, Wnt signaling and many more . Recent Primary cilia dysfunction plays a fundamental role in numerous hereditary organ-specific or syndromic diseases that are collectively called ciliopathies. Diseases and syndromes resulting from pathological changes in the primary cilium are diverse and occur in their entirety relatively frequent (1:2000) . SimilarThe relationships between primary cilia and proteostasis as well as between primary cilia and energy homeostasis are emerging fields in the scientific community and mechanisms that mediate primary cilia or basal body derived signals to modulate both processes are largely unknown. To bring together our current knowledge in the field, we present a compilation of four excellent review articles and two original research articles that contribute to shed light into this fascinating Research Topic.Aslanyan et al. deploys a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling to fill this gap and to create an overview of proteins modified with ubiquitin within the primary cilium. The authors identify several key proteins involved in signaling, cytoskeletal remodeling and protein trafficking including ESCRT-dependent clathrin-mediated endocytosis-related proteins and structural components of caveolae, e.g., CAV1, CAVIN1, EHD2.Ubiquitination plays a crucial role in promoting substrate unfolding and subsequent degradation by the proteasome, making it the primary inducer of this process. However, to this end a comprehensive landscape of ubiquitinated proteins at the cilium is missing. Barbeito et al. provide evidence, that ubiquitination of the complex could play a role in the regulation of complex stability. In line with this, the authors provide a list of EVC interacting proteins that includes the deubiquitinating enzyme USP7, suggesting that USP7 functions at the EVC-EVC2 complex. Furthermore, the authors identify previously unknown targeting signals in the C-terminus of EVC2 that are essential for the accumulation of the complex at the ciliary base.Two specific ciliopathies, Ellis van Creveld syndrome (EvC) and Weyers acrofacial dysostosis (WAD), primarily affect skeletal development. Both are caused by mutations in genes encoding for proteins of the heterodimeric, ciliary transmembrane EVC-EVC2 complex that is involved in Hedgehog signaling. Despite the significance of this complex, it remains only poorly understood how the stability and targeting to the place of action within the cilium is mechanistically controlled. Scamfer et al. provide an overview of the current knowledge regarding regulation of energy homeostasis by the cilium in adipose tissue and regarding ciliary proteins taking part in hypothalamic neuron signaling and adipocyte differentiation.Bardet-Biedl Syndrome  and Alström Syndrome , have been linked to obesity and metabolic disorders. Recent studies emphasize the significant role of the primary cilium in maintaining whole-body energy homeostasis. This is achieved through ciliary signaling in various cell types and tissues relevant to metabolism. In a comprehensive review, Melena and Hughes the current understanding of how cilia contribute to islet hormone regulation and glucose homeostasis is consolidated. People with diabetes of all types experience glucose dysregulation due to changes in the function and coordination of pancreatic islet cells. Various events leading to hormone secretion to regulate these processes are precisely regulated at/by the primary cilium, among them ciliary pathways that govern insulin exocytosis and intercellular communication.In a comprehensive review by Brewer et al. provide a brief overview of syndromic ciliopathies and monogenic cilia signaling mutations related to obesity and focus on neuronal cilia to underscore the critical role of neuronal cilia-mediated signaling in maintaining proper energy homeostasis. Furthermore, the authors summarize literature on cilia and leptin-melanocortin signaling, as well as on changes in ciliary G protein-coupled receptor (GPCR) signaling and put a focus on brain regions where cilia are involved in energy homeostasis.Claude-Taupin et al. explore the intricate cross-talk between autophagy, the primary cilium, and physical forces in the regulation of processes like mitochondrial biogenesis and lipophagy. Recent developments summarized in this review article unveil an interconnection between autophagy and the primary cilium, which affects autophagy but also contributes to the control of ciliogenesis.In conclusion, this Research Topic includes six articles addressing novel and updated aspects related to the regulation of proteostasis and energy homeostasis at/by the primary cilium and will likely turn into a standard reference in the coming years.


Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K–Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor–PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism ( Akt in turn phosphorylates tuberous sclerosis complex 2 (TSC2) to inhibit TSC function by inducing its dissociation from lysosomal membranes, where the mTORC1-activating Rheb GTPase resides –Akt pathway , 12. Rec resides , 14, 15. resides , 17, 18. resides , 20, 21. resides . The reg changes . A well- changes . While n changes , 25, 26.e.g., the GATOR2, GATOR1, and Ragulator–LAMTOR complexes), which converge on heterodimeric Rag GTPases (Rag A or B bound to Rag C or D) tethered to lysosomal membranes. Amino acid stimulation of amino acid–starved cells increases GTP loading of RagA or RagB, which recruits mTORC1 to lysosomal membranes through its Raptor subunit , and the readdition of amino acids induces the rapid dissociation of TSC from lysosomal membranes  also recruit TSC to lysosomes to inhibit mTORC1  and likely other translation-related factors   , 34.i.e., elevated AMP or ADP), phosphorylates mTOR and Rictor to activate mTORC2 directly in response to energetic stress, which suppresses apoptosis . Our subsequent work demonstrated that another type of cell stress, that is, elevated intracellular pH (pHi), increases AMPK activity to promote mTORC2 signaling in cooperation with an unknown input to suppress apoptosis during growth factor limitation  at an alkaline pH 8.3 increased the phosphorylation of Akt S473 but not S6K1 T389 in a manner sensitive to Torin1 (an ATP-competitive mTOR inhibitor) . Thus, an alkaline pHe is sufficient to increase mTORC2 but not mTORC1 signaling in the presence of growth factors. It is important to note that Demetriades et al.  had no effect on mTORC1 signaling to S6K1 in MEFs. To better understand this finding, we found that inhibition of class I PI3Kα with the drug BYL719  blocked the increase in Akt P-S473 mediated by alkaline pHe in CM . As growth factors represent a strong activating input to mTORC1, we reasoned that we should analyze serum-starved MEFs in order to probe a potential role for alkaline pH in the regulation of mTORC1. Refeeding serum-starved immortalized  or primary MEFs  with serum-free DMEM at pH 8.3 for 5 to 60 min increased the phosphorylation of several mTORC1 substrates, that is, S6K1-T389, 4EBP1-S65, and ULK1-S757, relative to MEFs refed with serum-free DMEM at physiological pH 7.4. Importantly, Torin1 ablated the pHi-induced phosphorylation of these targets (B and C). These results demonstrate that an alkaline pHe is sufficient to increase mTORC1 signaling in the absence of serum growth factors. In these experiments, we noted that serum-free media (SFM) at alkaline pH also increased the phosphorylation of Akt T308 (a direct target of PI3K-dependent PDK1) and Akt S473 (a direct target of mTORC2) (A–C). Consistent with mTORC2-mediated Akt S473 phosphorylation stabilizing Akt T308 phosphorylation (A–C). We also found that refeeding serum-starved MEFs with media adjusted to increasingly basic or acidic pH values increased  or decreased  mTORC1 and mTORC2 signaling, respectively. Finally, we analyzed how SFM at alkaline pH controls mTORC1 and mTORC2 signaling in other serum-starved cell lines, including C2C12, U2OS, HeLa, and human embryonic kidney 293 (HEK293) cells (A–D). In C2C12 and U2OS cells, SFM at an alkaline pH increased both mTORC1 (S6K1 P-T389) and mTORC2 (Akt P-S473) signaling (A and B). In serum-starved HEK293 cells, SFM at an alkaline pH increased mTORC1 signaling, albeit modestly, and mTORC2 signaling (C), whereas in serum-starved HeLa cells, SFM at an alkaline pH increased mTORC1 but not mTORC2 signaling (D). Taken together, these results indicate that media at an alkaline pH increases mTORC1 and/or mTORC2 signaling in several cell lines, including primary MEFs, during growth factor limitation, albeit to different degrees for reasons unclear at this time.While investigating the activation of the AMPK–mTORC2 axis by alkaline pHi in our prior work , we notehibitor)   cells , A–D. Inignaling , A and Bignaling C, whereaignaling D. Taken green cells) relative to those maintained in media at physiological pH (blue cells) (A and B). These results demonstrate that shifting cells to SFM at alkaline pH leads to a rapid rise in pHi.Prior work by other groups and us demonstrated that refeeding cells with serum-containing CM at an alkaline pH raises pHi rapidly , 48, 49.e cells) , A and B4Cl). NH4Cl increases cytosolic pH upon rapid diffusion of NH3 (a weak base) into the cell and conversion to ammonium ion (NH4+) by scavenging protons from the cytosol, thus increasing cytosolic pH , as measured microscopically with cSNARF1-AM. Biochemically, treatment of serum-starved MEFs with NH4Cl increased mTORC1 signaling weakly (S6K1 P-T389) but increased PI3K (Akt P-T308) and mTORC2 (Akt P-S473) signaling strongly (E).As an alternative method to raising pHi, we used ammonium chloride  antiporter on the plasma membrane that pumps hydrogen ions (H+) from the cytosol to the extracellular space. Thus, cariporide decreases pHi by blunting H+ efflux (F). These results indicate that attenuating H+ efflux from the cell suppresses mTORC1 and mTORC2 signaling likely by acidifying the cytosol, thus countering the alkalizing effect of media at pH 8.3. Note that NHEI represents one of many hydrogen ion transporters in cells, thus explaining why cariporide only partly blunts the activation of mTORC1 signaling in response to alkaline pHi. Taken together, these results indicate that altering pHi by three independent methods  modulates mTORC1 and mTORC2 signaling in the absence of serum growth factors, with alkaline pHi or acidic pHi increasing or decreasing mTORC1/2 signaling, respectively.We next sought to confirm that culturing cells in SFM at an alkaline pH increases mTORC1 and mTORC2 signaling by raising pHi. To do so, we used the drug cariporide (a more-specific derivative of amiloride) to reduce pHi pharmacologically. Cariporide inhibits NHE1 (sodium-hydrogen exchanger isoform 1), a sodium–proton  each increased S6K1 P-T389, whereas the addition of both together failed to increase S6K1 P-T389 further (A). When we stimulated cells with a submaximal dose of insulin (5 nM), however, alkaline pHi and insulin increased S6K1 P-T389 in an additive manner (B). Taken together, these data are consistent with the idea that alkaline pHi mimics growth factor signaling. As insulin and growth factors were shown several decades ago to increase pHi in fibroblasts and frog skeletal muscle by activating NHEI .Growth factors and amino acids activate mTORC1 cooperatively through parallel pathways, although some crosstalk occurs. We, thus, next investigated the relationship between mTORC1 signaling induced by alkaline pHi  further A. When we manner B. Taken ing NHEI , 60, 61, however C.Figure versus amino acids. We first tested how the addition of the commercial amino acid solution adjusted to pH 7.4 increases mTORC1 signaling relative to the addition of the original, unadjusted pH ∼10 commercial amino acid solution. As expected, after starving MEFs of both growth factors and amino acids, amino acids at pH 7.4 increased S6K1 P-T389 but not Akt P-S473; amino acids at pH ∼10 increased S6K1 P-S6K1 much more strongly, however, and also increased Akt P-S473 (D). These results suggest that alkaline pHi and amino acid signaling increase mTORC1 signaling additively by parallel pathways. Moreover, they demonstrate that alkaline pHi but not amino acids increase mTORC2 signaling. To analyze the effects of amino acids versus an alkaline pHi on mTORC1 signaling more carefully, we tested their ability alone and together to increase mTORC1 signaling. Refeeding serum- and amino acid-starved MEFs with media at pH 8.3 in the presence of amino acids increased mTORC1 signaling to a greater extent than either stimulus alone (E) (lane 4 versus lanes 2 and 3). These results suggest that amino acids and alkaline pHi signal in parallel to promote mTORC1 signaling. Interestingly, amino acid stimulation attenuated the ability of an alkaline pHi to increase in Akt P-T308 and P-S473 (lane 4) (E). Perhaps the well-established ability of mTORC1 to mediate negative feedback on PI3K flux  E. PerhapI3K flux , 63, 64 contexts , 66.A). BYL719 also blocked the PDK1-dependent phosphorylation of the activation loop sites on S6K1 (P-T229) and Akt (P-T308) as well as the phosphorylation of the Akt substrates TSC2-S939 and proline-rich Akt substrate of 40 kDa (PRAS40)-T246 (A). The Akt inhibitor MK2206 blocked the ability of alkaline pHi to increase the phosphorylation of S6K1-T389, 4EBP1-S65, Akt-T308, TSC2-S939, and PRAS40-T246 (B), thus phenocopying the effect of PI3K inhibition. Collectively, these results indicate that PI3K–Akt signaling is required for the activation of mTORC1 and mTORC2 signaling by alkaline pHi in the absence of serum growth factors.To ask whether the ability of alkaline pHi to increase mTORC1 signaling requires PI3K, we treated serum-starved MEFs with BYL719 prior to incubation in SFM at pH 8.3. BYL719 blocked the ability of alkaline pHi to increase the phosphorylation of S6K1-T389, 4EBP1-S65, and Akt-S473 A. BYL71940)-T246 A. The AkS40-T246 B, thus pA–C and A and B). It was therefore important to determine whether the ability of alkaline pHi to increase mTORC1 signaling occurs independently of the activating effect of alkaline pHi on mTORC2 signaling or not. We thus studied Rictor−/− MEFs stably expressing either vector control or rescued with hemagglutinin (HA)-tagged Rictor. Restoring mTORC2 function boosted S6K1 P-T389 overall in rescued Rictor−/− MEFs refed with SFM at either pH 7.4 or 8.3 (C) . In Rictor−/− MEFs expressing vector control (V) and lacking mTORC2-dependent Akt P-S473, alkaline pHi still increased PI3K signaling to Akt (P-T308), TSC2 (P-S939), and PRAS40 (P-T246) as well as mTORC1 signaling to S6K1 (P-T389) (C). In this context, Torin1 had no inhibitory effect on Akt P-T308 or TSC2 P-S939 (C). In Rictor−/− (V) MEFs, BYL719 blocked the ability of media at pH 8.3 to increase S6K1 P-T389 and Akt P-T308 (D). Collectively, these results demonstrate that alkaline pHi increases the PI3K-dependent activation of Akt and mTORC1 in the absence of mTORC2 function.Phosphorylation of Akt S473 by mTORC2 often increases Akt T308 phosphorylation, possibly by recruiting PDK1 and/or stabilizing Akt T308 phosphorylation , 47, whe4 or 8.3 C (likely(P-T389) C. In thi2 P-S939 C. In Rict P-T308 D. CollecPIK3Cα, which encodes the catalytic p110 subunit of class I PI3Kα, promote the growth of many cancers including breast cancer because of elevated PI3K activity, we tested whether breast cancer MCF7 cells bearing an oncogenic mutation in PIK3CA (E545K) would be resistant to the ability of an alkaline pHi to increase mTORC1 and/or mTORC2 signaling. As expected, serum-starved MCF7 cells displayed an easily detectable level of BYL719-sensitive mTORC1 (S6K1 P-T389) and mTORC2 (Akt P-S473) signaling because of elevated PI3K activity. In this context, an alkaline pHi failed to increase mTORC1 or mTORC2 signaling . As expected, insulin stimulation of serum-starved MEFs at physiological pH reduced the colocalization of TSC2 with LAMP1, as reported previously (A and B) (B). As the ability of alkaline pHi to dissociate TSC2 from lysosomes and increase mTORC1 activity and signaling was PI3K dependent, we next asked whether alkaline pHi increases PI3K activity. We refed serum-starved MEFs with SFM at pH 7.4 or pH 8.3 and measured the levels of PIP3 in purified lipids by an ELISA. As a positive control, we stimulated cells in media at pH 7.4 with insulin. Media at pH 8.3 increased PIP3 levels relative to media at pH 7.4 in a statistically significant and BYL719-sensitive manner (C and D). As expected, insulin increased PIP3 levels. These results indicate that alkaline pHi is sufficient to increase PI3K activity in the absence of growth factors. Taken together, they reveal that by increasing PI3K activity and Akt signaling, alkaline pHi dissociates TSC2 from the lysosomal surface, thus enabling Rheb to increase mTORC1 catalytic activity.We next sought to better define the mechanism by which alkaline pHi increases mTORC1 signaling. As activation of Akt by PI3K dissociates the TSC complex from lysosomal membranes through Akt-mediated inhibitory phosphorylation of TSC2 , we askeeviously , A and BA and B) . Thus, aA and B) . RefeediA and B) B. As thee manner , C and D7GTP-coupled Sepharose beads to assess the ability of alkaline pHi to dissociate 4EBP1 from eIF4E through mTORC1-mediated 4EBP1 phosphorylation. After serum starvation, refeeding MEFs with serum-free DMEM at pH 8.3 was sufficient to increase 4EBP1 phosphorylation and dissociate 4EBP1 from eIF4E, which occurred in a Torin1-sensitive manner (A). Pretreating cells with cariporide to suppress H+ efflux and thus reduce pHi blunted the ability of alkaline pHi to dissociate 4EBP1 from eIF4E. As the pH of DMEM at pH 8.3 falls quickly because of the strong buffering capacity of DMEM incubated in an atmosphere of 7.5% CO2, we incubated cells at low CO2  to maintain an elevated pH for a longer period. This modification to our method for raising pHi enabled us to study protein synthesis (see later), a process requiring a longer amount of time. In cells cultured at low CO2, alkaline pHi dissociated 4EBP1 from eIF4E and increased the association of eIF4G with eIF4E in a statistically significant and PI3K- and mTOR- dependent manner (B and C). As in other experiments, alkaline pHi increased the PI3K- and mTOR-dependent phosphorylation of 4EBP1 and S6K1 in cells cultured at low CO2 in this context (B). These results indicate that elevated pHi inhibits 4EBP1 function and promotes the assembly of translation preinitiation complexes.As protein synthesis represents a major cellular function promoted by mTORC1 , 34, we e manner A. Pretret manner , B and C context B. These 2  or low CO2  with serum-free DMEM at pH 7.4 or pH 8.3 for 60 min that lacked or contained a pulse of puromycin for the last 10 min, we found that alkaline pHi was sufficient to increase protein synthesis in a statistically significant manner at low CO2, which was PI3K and mTOR dependent (D and E). As expected, stimulation of serum-starved cells cultured at physiological pH 7.4 with insulin also increased protein synthesis (D and E). Consistently, alkaline pHi increased the PI3K- and mTOR-dependent phosphorylation of 4EBP1 and S6K1 T389 in this experimental context.We next asked whether increased assembly of eIF4E-based translation preinitiation complexes leads to increased protein synthesis. To do so, we employed the nonradioactive SUnSET assay, which measures the incorporation of the tRNA-mimetic compound puromycin into nascent polypeptide chains by simple Western blotting using an antipuromycin antibody . Upon reependent , D and Eynthesis , D and Ei.e., DMEM containing FBS) (lane 1) or in SFM  (lane 2) overnight (16 h) and then refed the cells for 1 h with either CM (lane 1) or SFM at pH 7.4 or pH 8.3 (lanes 2–7). As expected, cells cultured in SFM at pH 7.4 for the full 17 h displayed increased apoptosis (lane 2) relative to those kept in CM for 17 h (lane 1), as monitored by Western blotting of cleaved caspase 3 (cCasp3) and cleaved poly(ADP-ribose) polymerase (cPARP), well-established apoptotic markers (A and B). Culturing MEFs in SFM at pH 8.3 and 7.5% CO2 reduced levels of cCasp3 and cPARP slightly (lane 3). Culturing MEFs in SFM at pH 8.3 and 0.1% CO2, however, reduced the levels of cCasp3 and cPARP significantly (lane 4) relative to physiological pH 7.4 (lane 2) and did so in a BYL719- and Torin1-sensitive manner (lanes 5 and 7) (A and B). While the mTORC1-specific inhibitor rapamycin blunted the ability of alkaline pHi to suppress apoptosis modestly (lane 6), the effect was not statistically significant (B). Consistent with other experiments, alkaline pHi increased PI3K signaling (Akt P-T308), mTORC1 signaling (S6K1 P-T389), and mTORC2 signaling (Akt P-S473) in a PI3K- and mTOR-dependent manner in this experimental context (A). Collectively, these results indicate that alkaline pHi protects against apoptosis during growth factor limitation by increasing PI3K–mTOR signaling.Cell survival represents an important cellular function mediated by mTORC2 and Akt , 12. Our markers , A and B5 and 7) , A and Bnificant B. Consis context A. CollecRictor−/− MEFs stably expressing either vector control (V) or rescued with HA-Rictor. Upon growth factor withdrawal, Rictor−/− (V) MEFs displayed higher overall levels of cCasp3 and cPARP than rescued Rictor−/− (HA-Rictor) MEFs (C) (lanes 2 versus 8). As expected, levels of Akt P-S473 were extremely low in Rictor−/− (V) MEFs, and reconstitution with HA-Rictor rescued Akt S473 phosphorylation and boosted Akt T308 phosphorylation with little effect on S6K1 T389 phosphorylation (C). In Rictor−/− (V) MEFs cultured in SFM at low CO2 (0.1%), alkaline pHi 8.3 increased S6K1 P-T389 and reduced levels of cCasp3 and cPARP relative to cells cultured at physiological pHi 7.4 (lane 4 versus lane 2). These effects were both rapamycin and Torin1 sensitive (lanes 5 and 6 versus lane 4), with Torin1 having a stronger effect than rapamycin (C). These results suggest that in the absence of mTORC2 function, increased mTORC1 signaling in growth factor–starved cells contributes to the evasion of apoptosis mediated by alkaline pHi. Collectively, we propose that activation of both mTORC1 and mTORC2 by alkaline pH promotes cell survival during growth factor withdrawal, with mTORC2 playing a more major role in cell survival than mTORC1.We next asked whether PI3K–mTORC1 signaling induced by alkaline pHi protects against apoptosis during growth factor deprivation in the absence of confounding mTORC2 survival signaling. As before, we analyzed or) MEFs C (lanes rylation C. In Ricapamycin C. These +) dynamics at the plasma membrane and possibly internal membranes (+) flux drive diverse oncogenic cell behaviors, including cell proliferation, evasion from apoptosis, and cell migration through pathways and mechanisms that remain incompletely defined . Mechanistically, alkaline pHi increases PI3K activity and signaling to Akt, which leads to inhibitory phosphorylation of TSC2 and PRAS40 and dissociation of TSC from lysosomal membranes. These events enable Rheb to activate mTORC1 during growth factor limitation. Activation of PI3K by pHi explains why the activation of mTORC2 by alkaline pHi was only partially dependent on AMPK, as reported in our prior work . Functionally, alkaline pHi increases initiation of cap-dependent translation in a PI3K- and mTOR-dependent manner in the absence of growth factors through inhibitory phosphorylation of 4EBP1, thus enabling assembly of the eIF4F translation preinitiation complex . Alkaline pHi also protects against apoptosis caused by the withdrawal of growth factors in a PI3K- and mTOR-dependent manner.This study demonstrates that an alkaline pHi mimics growth factor–PI3K signaling to mTORC1 and mTORC2, pathways hyperactivated in many human tumors that contribute to their aberrant growth, proliferation, and survival , 71. We Fig. 7ior work . By mimiet al.  that promote glycolytic and pentose phosphate carbon flux. Thus, an alkaline pHi appears to influence metabolic adaptation as well as classical oncogenic cell behaviors.Cancer cells often bear an alkaline pHi through the upregulated expression of transporters that actively extrude cytosolic acid , 44, 45. in vivo . We spec in vivo . Interes  as a waste byproduct. Merhi et al.  but increased PI3K (PRAS40 P-T246) and mTORC2 (Akt P-S473) signaling strongly. In addition, NH4Cl increased the proliferation of cells deprived of glutamine  increased S6K1 phosphorylation and attenuated apoptosis concomitantly in a rapamycin-sensitive manner relative to physiological pH and normal CO2  during growth factor withdrawal. It is important to note that as some mTORC1 substrates display only partial sensitivity to rapamycin   , 78, a r have been documented in the epidermal growth factor receptor, the tumor suppressor protein p53, Ras-GRP1, and β-catenin, which alters their activities in cancer cells bearing an alkaline pHi and contributes to the transformed behaviors of the cells bearing these mutations  bearing charge-changing Arg to His mutations  through dynamic protonation and deprotonation, a post-translational modification akin to phosphorylation, ubiquitination, acetylation, and so forth . In factutations , 81, 82.utations , 80, 81.Collectively, these findings reveal that pHi functions as an internal rheostat that fine-tunes the level of PI3K–mTOR signaling, likely in response to physiological and pathological cues. In the context of cancer, we propose that elevated PI3K–mTORC1 and/or PI3K–mTORC2 signaling may contribute to the growth, proliferation, and survival of cancer cells bearing an alkaline cytosolic pH in growing tumors deprived of essential growth factors, prior to the onset of angiogenesis. As a final note, this study provides a cautionary tale highlighting the importance of controlling pH appropriately in cell cultures, as inadvertent alteration of the pHe affects pHi, cell signaling, and cell function .4Cl was from ThermoFisher . cSNARF1-AM was from Invitrogen . Cariporide was from MilliporeSigma .General chemicals were from ThermoFisher Scientific or MilliporeSigma. Protein A-Sepharose beads were from GE Healthcare . NP40, Brij35, and CHAPS detergents were from Pierce. Complete Protease Inhibitor Cocktail (EDTA-free) tablets was from MilliporeSigma . 7-methyl-GTP-Sepharose 4B beads were from GE Healthcare . Immobilon-P polyvinylidene difluoride (PVDF) membrane (0.45 μM) was from MilliporeSigma. Reagents for enhanced chemiluminescence were from either Alkali Scientific  or Advansta (WesternBright Sirius horseradish peroxidase [HRP] substrate). Torin1 was a gift from Dr David Sabatini. Rapamycin was from Calbiochem . BYL719 was from Selleck . MK2206 was from Selleck . NHThe following antibodies were from Cell Signaling Technology: Akt P-S473 , Akt P-T308 , Akt P-T450 , Akt , mTOR , Raptor , S6K1 P-T389 , 4EBP1 P-S65 , 4EBP1 P-T70 , 4EBP1 , eIF4E , eIF4G , PRAS40 P-S246 , PRAS40 , TSC2 P-S939 , TSC2 , ULK1 P-S757 , ULK1 , cCasp3 , cleaved PARP , and α-tubulin . mTOR P-S2481 was from MilliporeSigma . The antipuromycin antibody was from MilliporeSigma . The S6K1 P-T229 antibody was from Abcam . Antipeptide polyclonal antibodies generated against Rictor  and S6K1  were custom-made with the aid of Covance, as described . Donkey 2. To serum-starve cells, cells were washed once in DMEM containing 20 mM Hepes (pH 7.2) and then refed with this medium overnight (∼16 h). To amino acid–starve cells, cells were washed once in PBS containing Ca2+ and Mg+ and then refed with amino acid-free DMEM  lacking or containing dialyzed FBS (10%)  for 50 min. After amino acid deprivation, cells were stimulated with amino acids by adding an amino acid solution  supplemented with l-glutamine  to a final concentration of (1×) (roughly the concentration of amino acids found in RPMI media). The pH of the commercial amino acid solution (∼ pH 10) was adjusted to physiological pH 7.4 with 1 N HCl prior to addition to cells. To culture cells at an alkaline pHe, and thus to raise pHi, the pH of the DMEM was adjusted to pH 8.3 (or the pH indicated) using NaOH. To maintain DMEM at an alkaline pH  for a longer period, cells cultured in DMEM (pH 8.3) were incubated at 37 °C in a humidified atmosphere set to low CO2 (0.1%). Where indicated, cells were pretreated with various drugs or chemicals for 30 min: Torin1 (100 nM), BYL719 (10 μM), MK2206 (10 μM), cariporide (10 μM), and NH4Cl (10 mM).Cell lines  were cultured in DMEM containing high glucose (4.5 g/l), glutamine (584 mg/l), and sodium pyruvate (110 mg/l)  supplemented with 10% FBS  and incubated at 37 °C in a humidified atmosphere with 7.5% COUnless indicated otherwise, cells were washed twice with ice-cold PBS, lysed in ice-cold buffer containing NP-40 (0.5%) and Brij35 (0.1%), and incubated on ice (15 min), as described . To mainescribed . Immunobescribed  and incuPrimary MEFs were isolated from day 12.5 mouse embryos, as described , 85.Rictor−/− MEFs stably expressing vector control (pPPM) or HA-Rictor were generated by lentiviral transduction. The HA-tagged Rictor complementary DNA was subcloned into a modified lentiviral vector, pHAGE-Puro-MCS (pPPM)   . MEFs were cultured on 15 cm plates and treated with media at pH 7.4 or 8.3, drugs, and/or insulin. The media were aspirated, 10 ml of ice-cold 0.5 M TCA was added, and the plate was incubated on ice for 5 min. Cells were scraped and collected into a 15 ml tube on ice and centrifuged at 950g for 7 min at 4 °C. The pellet was resuspended in 3 ml of 5% tricarboxylic acid/1 mM EDTA, vortexed, centrifuged at 3000 rpm for 5 min, and washed once again in 3 ml 5% trichloroacetic acid/1 mM EDTA. Neutral lipids were next extracted by adding 3 ml of MeOH:CHCl3 (2:1) to the pellet and vortexing 10 min followed by centrifugation at 950g for 5 min. The supernatant was discarded, and this extraction step was repeated once more. To extract acidic lipids, 2.25 ml of MeOH:CHCl3:HCl (12 N) (80:40:1) was added, and the solution was vortexed continuously for 25 min. Extracts were centrifuged at 950g for 5 min, and the supernatant was transferred to a new 15 ml tube. About 0.75 ml of CHCl3 and 1.35 ml of 0.1 M HCl was added to the supernatant, vortexed, and centrifuged at 950g for 5 min to separate the lower organic and upper aqueous phases. The organic phase (∼1.45 ml) was transferred to a new tube for the measurement of PIP3. All samples were dried in a vacuum dryer for 1 h. PIP3 samples were resuspended in 140 ml of PBS–Tween +3% Protein Stabilizer (provided in the Echelon kit). Samples were sonicated in an ice-water bath for 10 min and spun down. The experiment was performed four times with technical duplicates. PIP3 levels were measured by ELISA, according to the manufacturer’s instructions .Levels of PIP2 (7.5%). Images were acquired using a 40× oil objective  at a resolution of 1024 × 1024 and an optical thickness of 1.18 μM . The cSNARF1-AM signal was excited with an argon laser at 488 nm, and images sets were collected simultaneously in two emission bandpass filters at 553 to 618 nm (for 580 nm) and 663 to 738 nm (for 640 nm). Nikon Elements software was used to create and pseudo-color the 640:580 nm ratiometric images with values ranging from 0 (violet) to 2.0 (red). MetaMorph software  was used to quantify the ratiometric images by measuring the average ratio of a region of interest within one cell, and 25 cells per image were quantified. A change in the 640:580 nm ratio and accompanying pseudo-colored image reflects a change in pHi.MEFs were cultured on 8-well cover glass bottom chambers  and loaded with cSNARF1-AM (10 μM)  for 30 min at 37 °C in serum-free DMEM (pH 7.4). The cells were then rinsed with serum-free DMEM (pH 7.4) and refed with this medium. Live-cell imaging (0–10 min) of cells cultured in serum-free DMEM (pH 7.4 or 8.3) was performed using a Nikon A1 confocal microscope equipped with a stage-top incubator that maintains temperature (37 °C) and CO2+ and Mg2+  and fixed with 3.7% formaldehyde for 10 min. Cells were then washed twice with PBS+, permeabilized in 0.2% TX-100 for 5 min, washed twice with PBS+, and blocked in 0.2% fish skin gelatin (FSG) for 1 h. Cover slips were inverted on primary antibodies (1:100 dilution)  diluted in PBS+ containing 0.2% FSG and incubated overnight. Cells were washed three times with PBS+, and cover slips were inverted on Alexa 488-conjugated anti-rabbit antibody (1:500 dilution)  or Alexa 594-conjugated antirat antibody (1:500 dilution)  diluted in PBS+/0.2% FSG, incubated for 1 h, and washed three times with PBS+. Cover slips were mounted on a slide using Prolong Gold with 4′,6-diamidino-2-phenylindole (Invitrogen). Images were captured using a 40× objective lens on a Zeiss LSM800 Confocal Laser Scanning Microscope, and images were analyzed using Fiji.MEFs were seeded on glass coverslips in 6-well plates and serum-starved overnight (∼16 h). Following various treatments, cells were washed with PBS containing Cai.e., pH 8.3) for a longer period, the cells were incubated at 37 °C in a humidified atmosphere at low CO2 . Puromycin (10 μg/ml) was added for the last 10 min of refeeding. Cells were lysed with ice-cold lysis buffer containing NP-40 (0.5%) and Brij35 (0.1%), and whole-cell lysates were immunoblotted with an antipuromycin antibody. Puromycin incorporation was analyzed using ImageJ  software. Equal protein loading of SDS-PAGE gels was confirmed by staining with SimplyBlue SafeStain , according to the manufacturer’s instructions.Protein synthesis was measured by SUnSET assay, as described . BrieflyAdobe Photoshop was used for image preparation, using only the levels, brightness, and contrast controllers equivalently over the entire image.p < 0.05 were considered significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.00001.Results are presented as mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test. Values of dfingar@umich.edu) and/or Dubek Kazyken, PhD (dkazyken@umich.edu). This article also contains The data shown in This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Tooth root development involves intricate spatiotemporal cellular dynamics and molecular regulation. The initiation of Hertwig’s epithelial root sheath (HERS) induces odontoblast differentiation and the subsequent radicular dentin deposition. Precisely controlled signaling pathways modulate the behaviors of HERS and the fates of dental mesenchymal stem cells (DMSCs). Disruptions in these pathways lead to defects in root development, such as shortened roots and furcation abnormalities. Advances in dental stem cells, biomaterials, and bioprinting show immense promise for bioengineered tooth root regeneration. However, replicating the developmental intricacies of odontogenesis has not been resolved in clinical treatment and remains a major challenge in this field. Ongoing research focusing on the mechanisms of root development, advanced biomaterials, and manufacturing techniques will enable next-generation biological root regeneration that restores the physiological structure and function of the tooth root. This review summarizes recent discoveries in the underlying mechanisms governing root ontogeny and discusses some recent key findings in developing of new biologically based dental therapies. Consequently, the loss of teeth instigates multifaceted detriments to both oral and systemic health, underscoring the exigency for swift and efficacious remedial interventions.4Dental integrity stands as an essential cornerstone of overall physiological well-being, given that teeth assume pivotal functions in processes such as mastication, digestion, phonation, and self-perception.7 In this context, nascent paradigms rooted in biological tooth regeneration have surfaced, leveraging the tenets of stem cell-based bioengineering.8 These innovative approaches portend outcomes that are more congruent with the innate physiology, durability, and bioresponsive dynamics of natural tooth structure, thereby heralding a promising and forward-looking therapeutic avenue.9Presently, osseointegrated dental implants emerge as the prevailing clinical benchmark for ameliorating tooth absence. However, it is noteworthy that certain limitations are attendant, encompassing elevated prerequisites for bone volume and an absence of sensory biofeedback mechanisms.10 This intricate collaboration of molecular dialogs advances through discrete phases, beginning with the development of the tooth crown and terminating with the formation of the tooth root.A pivotal prerequisite for advancing the therapeutic trajectory is the elucidation of ontogenetic processes that underpin the morphogenesis of tooth roots. This pursuit holds the promise of unveiling novel focal points for inciting intrinsic dental repair mechanisms and the orchestrated construction of tooth organ substitutes. The orchestration of tooth organogenesis ensues through a sequence of orchestrated, reciprocal molecular interplays between distinct specialized cell populations-the dental epithelial cohort and the cranial neural crest (CNC)-derived mesenchymal contingent.11 Following the accomplishment of crown development, the root morphogenesis commences, spearheaded by the inception of Hertwig’s epithelial root sheath (HERS). The orchestration of the signaling cascades in the HERS serves as the conductor directing odontoblast differentiation, ultimately leading to the deposition of radicular dentin. Concurrently, the dental follicle takes on the role of generating cementoblasts, which oversee the deposition of acellular and cellular cementum onto the nascent root surface, subsequent to the dissolution of HERS.A strip of oral epithelium that has thickened marks the beginning of the creation of the crown, giving rise to the dental lamina.12 It is worth acknowledging that deviations from the norm within these signaling pathways choreographies underlie a panoply of root anomalies, spanning from abridged root dimensions to anomalies at the furcation junctures. Thoroughly deciphering the molecular mechanisms underlying the morphogenetic processes of tooth roots promises to unveil innovative perspectives for biotherapeutic interventions that activate intrinsic tooth repair and regeneration cascades.Throughout the trajectory of root ontogeny, the precise orchestration of spatiotemporally regulated signaling pathways assumes a nonpareil significance, serving as the compass directing HERS dynamics and the interplay between epithelial and mesenchymal compartments. Key protagonists in this narrative include signaling pathways entities, including Bone Morphogenetic Protein (BMP), Wnt, Transforming growth factor-β (TGF-β), Ectodysplasin A (EDA), and Sonic hedgehog (SHH), alongside a constellation of Mitogen‑activated protein kinase (MAPK) cascades.14 The crown develops first, marked by the thickened oral epithelium proliferates and penetrates into the beneath CNC-derived mesenchyme and then give rise to the dental lamina structure.15 The dental lamina undergoes rapid cell proliferation, morphological expansion, and elongation, eventually developing into the enamel organ.17 Simultaneously, the CNC-derived mesenchyme condenses surrounding the dental lamina, serves as the major source of DMSCs. The dental lamina, together with the condensed mesenchyme around it, forms the tooth germ. The dental lamina develops into the enamel organ, and meanwhile the condensed mesenchyme diverges into the dental papilla and the dental follicle at the cap stage.18 The enamel organ produces enamel, while the dental papilla and the dental follicle subsequently form pulp-dentin complex and periodontal tissues, respectively.21Tooth development involves the lineage development of CNC cells, complex mutual interactions between dental epithelium and dental mesenchymal stem cells (DMSCs), and a precise sequence of morphological stages.Tfap2b+/Lhx6+/Pax9+ in the condensed CNC-derived mesenchyme is identified as the progenitor cell population contributing to tooth formation. During the cap stage, the homogenous CNCCs segregate into dental papilla and dental follicle lineages, marked by specific genes like Crym/Egr3/Fgf3 and Epha3/Foxf1/Fxyd7 respectively.21 The dental papilla and follicle undergo further segregation at the bell stage. The dental papilla divides into distinct coronal and apical domains, marked by expression of Lmo1/Fgf3/Smpd3 and Lhx6/Fst/Gldn respectively. Concurrently, the dental follicle divides into Lepr/Foxf1/Bmp3 marked lateral domain and Aldh1a2/Rasl11a/Sgk1 marked apical domains. For instance, Lepr+ lateral follicle cells mainly form periodontal tissues. At the postnatal stage, dental papilla contains four distinct cell populations, including mature Phex+/Ifitm5+ odontoblasts, Enpp6/Fabp7+ coronal papilla, Nnat+/Rab3b+ middle papilla, and Aox3+/Tac1+ apical papilla cells. The latter was identified as bipotent progenitors for odontoblasts and pulp. In the follicle, Pthrp+/Bmp3+/Tnmd+ lateral and Pthrp+/Smoc2+/Slc1a3+ apical domains resemble those present at the bell stage and Pthrp+ dental follicle progenitors generate periodontal ligament (PDL) and cementum at the root-forming area and the furcation region.22 Particularly, a population of Slc1a3+ cells from the dental follicle’s apical area control cell lineages and aid in the formation of periodontal tissues at the root furcation. In addition, a range of other cells, such as glial cell derivatives, and pericytes can also serve as stem cell resources for dental mesenchyme.25 The perivascular niche contains NG2+, Gli1+, and Acta2+ cells that can differentiate into odontoblasts. The perineural niche is derived from Schwann cell precursors. Throughout the formation and regeneration following tooth damage, dental pulp cells and odontoblasts can be differentiated from PLP1+ and Sox10+ Schwann cells.26The tooth germ experiences three stages in sequence during development: bud, cap, and bell stage. During mouse molar development, CNC-derived DMSCs separate into various cell populations and domains, which is a crucial phase in DMSCs differentiation and functional specialization Fig. . At the 27 As the crown nears completion, the inner and outer enamel epithelium at the edge cervical loop continues to proliferate and elongate towards the future apical foramen from the cervical loop, which is located at the enamel organ’s cervical margin and is the region where the future crown and root of the tooth meet, also known as cementoenamel junction. This elongation gives rise to HERS, which is vital for the root development.28Gli1+ cells, residing in HERS, apical dental papilla, and dental follicle, have been identified as progenitor cells in the root and play a crucial role during root development.29. Moreover, Sox2+ stem cells that resides in dental epithelial contribute to all epithelial cell lineages at the bell stage, while Osx+ progenitor cells that distributes in dental papilla and dental follicle, especially in the area adjacent to HERS, differentiate into odontoblasts, dental pulp cells, cementoblasts, and PDL cells.32Sox2+ cells located at the cervical loop continue to exist in mouse incisors, while they disappear in mouse molars after birth.34 The cells from cervical loop and HERS, respectively, have their own different gene expression patterns. HERS cells have a relative higher level of BMP2 and BMP4, but a lower level the genes involved in enamel formation and mineralization, such as amelogenin (AMGN), ameloblastin (AMBN), osteopontin (OPN), bone sialoprotein (BSP), Osteocalcin (OCN) and dentin sialophosphoprotein (DSPP).35 HERS cells also express both Cytokeratin 14 (Ck14) and Epithelial cadherin (E-cadherin), while detached cells from HERS are only Ck14 positive with reduced E-cadherin expression.37Once the crown has formed during the late bell stage, the root development commences, marked by the formation of HERS.38 If the entire epithelial diaphragm boundary develops equally and ultimately merges, a normal single root will be formed. In the development of multirooted teeth, when the root is going to divide, several tongue-shaped epithelial diaphragms formed from HERS protrude toward each other and subsequently fuse to form continuous bridges, which will be the root furcation. The dental papilla is then divided into equivalent parts, ultimately resulting in the formation of equivalent roots.39At the initial phase of HERS development, the ends of the double-layered enamel epithelium of HERS curve toward the dental papilla, forming an epithelial diaphragm structure, histologically encapsulating the dental papilla and surrounded by the dental follicle. This diaphragm has a determinantal role in root morphology, including shape, number, and length of the root.41 As dentin formation progresses, the dental papilla becomes confined to a smaller space and eventually forms the dental pulp. Once the very first coat of radicular dentin is formed and the tooth root begins to elongate, HERS loses its integrity but remains a network of connections during root development through localized apoptosis or epithelial-to-mesenchymal transition (EMT), and the remaining HERS cells in the PDL are called ‘epithelial rests of Malassez (ERM)’, which contribute to cementum regeneration.44When the outermost mesenchymal cells of the apical papilla establish connections with the innermost cells of the HERS, they are induced to differentiate into odontoblasts and gradually retreat to the center of the dental papilla, secreting radicular dentin.45 In the region of the cervical root, cementoblasts first deposit cementum on the dentin and withdraw from the progressing mineralization front so that they do not incorporate themselves into the cementum, termed acellular cementum, also known as primary cementum. In more apical and inter-radicular areas, unlike acellular cementum formation, cellular cementum, also known as secondary cementum, is secreted by cementoblasts when the tooth reaches the occlusal plane, mineralizing rapidly and occasionally embedding some cementoblasts as cementocytes.49 The dental follicle also contributes osteoblasts and periodontal ligament cells to form the PDL and alveolar bone.50 In addition to dental follicle resources, some researchers have suggested that cementoblasts and PDL fibroblasts can also be derived from HERS cells through EMT and can be directly engaged in the deposition of cementum and PDL fibers during the development of periodontal tissues.51Following the disintegration of the HERS, the lateral mesenchymal cells of the dental follicle acquire interaction with the radicular dentin and then differentiate into cementoblasts, forming acellular cementum and cellular cementum.42 HERS development seems to be an especially solid procedure at the shift from the crown to the root morphological formation, whereas apical HERS development related to root elongation and the establishment of the root furcation appears to be more vulnerable to a variety of intrinsic as well as extrinsic negative effects.53. Developmental abnormalities in HERS can cause defects in tooth root formation.The HERS serves as a critical regulator in the proper formation of the tooth root. One primary role of it in tooth root formation is to induce odontoblast differentiation and subsequent dentinogenesis.53 Another phenomenon associated with failed HERS disintegration is the formation of enamel pearls. This occurs when HERS cells keep attached to the dentin and differentiate into ameloblasts, enamel pearls are formed.54When the continuity of HERS is compromised, stem cells from apical papilla (SCAPs) failed to differentiate into odontoblasts, leading to radicular dentin defects at that site and the formation of lateral accessory canals. Conversely, if HERS does not disintegrate after the first layer of dentin is formed and remains adhered to the dentin, it will block the engagement between dental follicle stem cells (DFSCs) and radicular dentin, preventing them from differentiating into cementoblasts and forming cementum. In some cases, delayed formation of the epithelial diaphragm in the multirooted teeth can result in taurodontism or single-rooted teeth. Following the completion of the bridges, future interradicular dentinogenesis and cementogenesis can be unaffected.14 Proper spatiotemporal coordination of these cell-cell interactions is mediated by several evolutionarily conserved signaling pathways.Root formation is susceptible to various effects that can influence molecular processes. The underlying molecular mechanisms tightly coordinate morphogenic events, cytodifferentiation, mineralization, and maturation during tooth root development in a precise spatial and temporal way. Any disruptions of the involved regulatory mechanisms could lead to root malformations. The interaction and regulation among the signaling pathways and a range of molecular factors contribute to various processes during tooth root development Fig. . Normal 55 Simultaneous conditional deletion of Bmp2 and Bmp4 in dental epithelium resulted in persistent HERS with reduced apoptosis and downregulation of BMP/SMAD and MAPK  signaling pathways in HERS cells. The impaired BMP/SMAD signaling pathway further caused decreased expression of SHH in HERS and downregulated expression of GLI family zinc finger 1 (GLI1) in root mesenchyme cells, leading to downregulation of the downstream target gene Nuclear Factor I C (NIFC). Reduced NIFC ultimately resulted in defective differentiation and maturation of root odontoblasts, decreased Osterix (OSX) and DSPP expression, and impaired dentin formation, resulting in short root anomaly. Dspp transgenic expression partially alleviated the short root phenotype.56 Therefore, optimal spatiotemporal expression levels of epithelial BMP ligands were required to properly coordinate HERS dissolution and odontogenesis through intricate reciprocal epithelial-mesenchymal molecular interactions over the entire course of root development.56 However, one study suggested BMP4 ligand was secreted by surrounding DPSCs and bound to BMPR-IB and BMPR-II expressed on HERS epithelial cells. Organ culture studies employing developing mouse molars demonstrated that BMP4 derived from mesenchyme bound to BMPR-IB/II dimers on HERS cells, thereby providing negative feedback to inhibit further HERS elongation while simultaneously promoting odontoblast differentiation.57 Mice harboring an epithelial-specific deletion of Smad4, a pivotal intracellular BMP effector, exhibited arrested root developmental owing to failed HERS elongation and stratification, confirming SMAD4’s indispensable role.58 Specifical deletion of Smad4 downregulated SHH expression within HERS and the surrounding inner enamel epithelium. Ectopic SHH expression in mutant mice partially rescued the root defects by restoring NFIC expression and normal dentin formation by newly differentiated odontoblasts.59 Epithelial SMAD4-SHH signaling pathway triggered mesenchymal NFIC expression to enable HERS morphogenesis and subsequent root formation. In contrast, conditional ablation of Smad4 specifically in dental epithelium led to prolonged molar crown morphogenesis associated with maintained cervical loop structures harboring proliferative Sox2+ epithelial stem cells. Additional deletion of Shh in the epithelium in these compound mutant mice partially rescued this aberrant phenotype and eliminated Sox2+ epithelial stem cells. Hence, BMP-SMAD4 signaling pathway in the mesenchyme provided crucial negative feedback to restrict dental epithelial stem cell expansion during the later phase of root formation by suppressing the SHH-GLI1 signaling pathway in the epithelium.33The BMP signaling cascade, particularly BMP2 was crucial in determining root patterning and elongation.β-catenin severely disrupted root morphogenesis and HERS dissociation. Studies suggested that suppressing β-catenin in HERS led to earlier disruption and thinner HERS, while stabilizing β-catenin in HERS prevented HERS dissociation after root dentin formation.60 Conditional deletion of β-catenin permitted molar tooth eruption accompanied by HERS elongation but completely abrogated root formation.61 Yet, odontoblast-specific deletion of β-catenin likewise caused premature pathological dissociation of HERS via upregulating BMP7, downregulating Noggin and Follistatin in odontoblasts, and accompanied by increased phosphorylation of SMAD1/5/8 in HERS cells.14 During molar furcation morphogenesis, canonical Wnt signaling pathway induced by epithelial WNT10A ligand critically patterned organized cell division and differentiation behaviors by modulating mesenchymal WNT4 expression and localized Wnt/β-catenin activity. In the furcation area, epithelial deletion of Wnt10a caused a decrease in epithelial cell proliferation but an increase in dental papilla mesenchymal cell proliferation, resulting in pathological taurodontism phenotype. Wnt10a deletion in dental epithelium causes compensatory upregulation of other Wnt ligands like WNT4, resulting in excessive β-catenin activation and overgrowth of dental papilla mesenchyme. Silencing of aberrant WNT4 partially rescued this abnormal furcation phenotype, validating that epithelial WNT10A precisely orchestrated dental epithelial and papilla mesenchymal cell proliferation by modulating the activation of mesenchymal WNT4 to guide root furcation morphogenesis.62The canonical Wnt/β-catenin signaling pathway was also indispensable for proper differentiation of functional odontoblast lineage cells and root dentin formation. During root development, β-catenin was essential for preserving the proliferation, polarity, and adhesion of HERS cells. HERS-specific deletion or overexpression of 63 During EMT, HERS cells must precisely balance antagonistic Sema-RhoA and TGF-β signaling pathways, whereby the Sema-RhoA signaling pathway promoted the maintenance of epithelial polarity and adhesion while TGF-β signaling pathway drove the acquisition of mesenchymal attributes and enhanced migratory capacity.36 Furthermore, mesenchymal TGF-β signaling pathway indirectly help sustain the residual dental epithelial stem cell population by modulating Wnt signaling pathway activity in cervical loop through the induction of key ligands like WNT5a and FGF3/1064.During the progression of tooth root maturation, HERS cells experienced EMT and relocated into the emerging periodontium. This EMT process could be induced in vitro by exogenous stimulation with either TGF-β1 or FGF2 growth factors, which activated MAPK/ERK intracellular signaling cascades.65 Eda-Edar signaling pathway represents another critical signaling pathway that directly participates in proper root formation and elongation. Both Ectodysplasin A (Eda) and Ectodysplasin A receptor (Edar) knockout mice exhibited substantially increased HERS proliferation and subsequent root developmental defects including shortened root length and taurodontism.66Moreover, DFSCs and cementoblasts could trigger apoptosis of both inner enamel epithelial-derived ameloblasts and HERS cells by upregulating FAS ligand (FASL) expression, which binds to and activates FAS-mediated extrinsic apoptotic signaling cascades.In summary, intricate molecular signaling networks centered upon BMP, canonical Wnt/β-catenin, TGF-β, Fas/FasL, and Eda pathways precisely directed HERS behaviors and epithelial-mesenchymal interactions at distinct developmental stage of tooth root morphogenesis. Comprehensive elucidation of these intersecting regulatory mechanisms using advanced genetic models will uncover novel therapeutic targets to enhance endogenous tooth root repair and inform sophisticated bioengineering strategies for whole tooth regeneration.67 SCAPs have a considerably greater proliferation and mineralization capacity than dental pulp stem cells (DPSCs).69 During tooth development, a substantial number of SCAPs reside in the apical papilla. Through complex molecular signaling pathways, the apical papilla provides a niche regulating the organized differentiation of SCAPs. To promote further root elongation and dentin formation, SCAPs can differentiate into odontoblasts.70 The regulation of biological behaviors of SCAPs, such as differentiation, maturation, and mineralization, is tightly controlled by a cascade of signaling pathways, including TGF-β, WNT, and MAPK pathways  express classic mesenchymal markers and have multi-differentiation potential.ays Fig. .71Fig. 3Alp and Ocn in a SMAD3-dependent manner. The TGF-β1’s inhibitory effect on SCAPs differentiation was enhanced by NFIC knockdown, whereas overexpression of NFIC antagonized these effects.72 Another study yielded the contrary results that TGF-β1 increased SCAPs proliferation and collagen production, which could be inhibited by blocking ALK5/SMAD2 and MEK/ERK pathways. Moreover, TGF-β1 had dual effects on regulation of Alkaline phosphatase (ALP) activity in SCAPs. For instance, low doses of TGF-β1 stimulated ALP activity which could be blocked by inhibiting ALK5/SMAD2 and MEK/ERK pathways, while higher doses inhibited ALP which could be rescued only by ALK5/Smad2 inhibition.73TGF-β1 inhibited SCAPs proliferation and osteogenic differentiation by downregulating osteogenic genes like β-catenin in odontoblasts, it was observed that odontoblast differentiation was impeded and the expression of DSPP, OCN, and Type I collagen (COL I) in regions anticipated to form the root was significantly diminished. The loss of differentiated odontoblasts prevented root dentin formation, mineralization, and periodontal attachment, though the epithelial root sheath developed normally but failed to induce odontoblast differentiation in the absence of β-catenin signaling pathway.74 Low-dose Wnt agonist LiCl inhibited Glycogen synthase kinase 3 beta (GSK3β) and β-catenin degradation in nuclear, leading to β-catenin accumulation and stimulating cell cycle genes, thereby enhancing SCAPs proliferation and differentiation.75 WNT3a and BMP9 were reported to synergistically promote the osteogenic/odontogenic differentiation of SCAPs, which required functional β-catenin.76 Apurinic/Apyrimidinic (AP) endonuclease 1 (APE1) expression decreased during tooth development. APE1 inhibition promoted osteogenic differentiation of DPSCs via stimulating the canonical Wnt signaling pathway.77 Similar results were obtained that Secreted frizzled-related protein 2 (SFRP2) blocked Wnt signaling pathway activation by competing with Frizzled receptor for Wnt, increasing β-catenin phosphorylation and nuclear exclusion and thus downregulating Wnt target genes. Sfrp2 overexpression enhanced while its knockdown inhibited osteogenic/odontogenic differentiation of SCAPs.79 LIM homeobox 6 (LHX6) coordinated Wnt activity and SCAPs differentiation to ensure correct root patterning and furcation development. LHX6 maintained active canonical Wnt signaling by inhibiting SFRP2 which physically interacted with WNT10A and suppressed WNT10A-induced odontoblast differentiation in the furcation area, resulting in defective dentin bridge formation. Moreover, LHX6 negatively regulated Cyclin-dependent kinase inhibitor 1C (CDKN1C) to maintain SCAPs proliferation.80 One recent study unraveled the tissue-specific role and molecular mechanisms of the non-canonical Wnt receptor, Receptor tyrosine kinase like orphan receptor 2 (ROR2), during molar root development. ROR2 was highly expressed in root-forming apical dental mesenchymal cells. Conditional deletion of Ror2 in Gli+ DMSCs impaired root elongation and furcation formation, inhibited dental mesenchymal cell proliferation, disrupted HERS invagination, and decreased odontoblast differentiation through epithelial-mesenchymal interactions, but without affecting crown morphogenesis. Mechanistically, ROR2 regulated dental mesenchymal cell proliferation and root patterning likely by modulating the activity of Cell division cycle 42 (CDC42).81 However, Wnt inhibition might be required for normal development, as β-catenin overexpression in dental mesenchyme resulted in upregulated SMAD4 in odontoblasts while downregulation of Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in cementoblasts, causing hypomineralized dentin and excessive cementum deposition respectively.83 Deletion of Runt-related transcription factor 2 (Runx2) in Gli1+ DMSCs resulted in impaired odontoblast differentiation, downregulation of NOTUM and upregulation of WNT/β-catenin signaling. RUNX2 directly upregulated the NOTUM expression, which was specifically expressed in pre-odontoblasts and inhibited HERS-secreted WNT ligands, WNT3a as well as WNT4, and thus regulated WNT/β-catenin signaling in the dental mesenchyme.84The proper development of roots and deposition of dentin and cementum rely heavily on the precisely spatiotemporal management of the Wnt signaling pathway. In the mice with specifically deleted Gli1+ DMSCs during root development. Arid1a deletion in Gli1+ DMSCs resulted in shortened roots and impaired dentin and periodontal tissues. ARID1A interacted with transcription co-Factor Pleomorphic adenoma gene-like 1 (PLAGL1) and directly regulated the transcription of GLI1, thereby controlling the switch from proliferation to differentiation.85 Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and ARID1A antagonistically regulated the expression of Cyclin dependent kinase inhibitor 2 A (CDKN2A) to control cell proliferation in the root-forming region, thereby coordinating furcation development and determining molar root number. Loss of EZH2 led to upregulation of CDKN2A, resulting in reduced cell proliferation and defective furcation formation. In contrast, monoallelic deletion of Arid1a rescued the root defects caused by loss of Ezh2.86At-rich interaction domain 1 A (ARID1A) was also expressed in 87 One study showed bFGF enhances stemness in SCAPs and decreases osteogenic/dentinogenic differentiation and related gene expression. Meanwhile, bFGF pretreatment enhanced the subsequent osteogenic differentiation potential of SCAPs.88 Epiregulin (EREG), an epidermal growth factor family member, stimulated the MEK/ERK and JNK signaling cascades, resulting in increased SCAPs proliferation.89Furthermore, growth factors played crucial roles in apical papilla development. SCAPs expressed all four fibroblast growth factor (FGF) receptors. Basic FGF (bFGF) can activate Extracellular signal regulated kinase (ERK) and Transforming growth factor beta-activated kinase 1 (TAK1) phosphorylation in SCAPs. The MEK/ERK inhibitor U0126 and the TAK1 inhibitor 5Z-7-oxozeaenol compromise bFGF-induced upregulation of Plasminogen Activator Inhibitor-1 (PAI-1), Urokinase-type Plasminogen Activator (uPA), Urokinase Plasminogen Activator Receptor (uPAR), and Tissue Inhibitor of MetalloProteinase-1 (TIMP-1) expression. They also partially reversed bFGF-mediated inhibition of OCN expression in SCAPs. This suggests bFGF may regulate SCAPs proliferation, migration, and matrix turnover while suppressing differentiation through MEK/ERK and TAK1 signaling pathways.90 17beta-estradiol (E2) enhanced SCAPs osteogenic/odontogenic differentiation without affecting proliferation, by upregulating ALP, DSPP, Dentin matrix acidic phosphoprotein 1 (DMP1), and matrix mineralization and activating JNK, p38 MAPK pathways and downstream nuclear factors like c-Jun and c-Fos.91 Estrogen receptor α (Erα) also enhanced SCAPs osteogenic/odontogenic differentiation via the same mechanism.92Additionally, lncRNA H19 promoted SCAPs osteogenic/odontogenic differentiation by binding to miR-141 and upregulating Sperm associated antigen 9 (SPAG9), which activated MAPK pathway by regulating the phosphorylation of p38 and JNK.29 Intriguingly, Lysine-specific demethylase 1A (KDM1A) had different regulatory roles at distinct stages during osteogenic/dentinogenesis. Inhibiting KDM1A reduced the activity of early osteogenic/dentinogenic markers and mineralization in SCAPs, but increased the expression of middle and late stage osteogenic/dentinogenic genes like Bsp, Dspp, and Dmp1. KDM1A can bind to Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) to form a protein complex. Inhibition of PLOD2 had similar effects as inhibition of KDM.93 Additionally, there was a study suggested inhibition of PI3K/Akt/mTOR signaling pathway enhanced osteogenic/dentinogenic differentiation and calcification of SCAPs and SCAPs spheroids.94 Discoidin domain receptor family member 2 (DDR2) expressed in dental papilla, odontoblasts, dental follicle, as well as PDL during tooth development. Mice lacking the Ddr2 gene exhibited short tooth roots, abnormal root/crown ratios, PDL defects, and progressive alveolar bone loss with impaired differentiation of cultured dental pulp and PDL cells.95 Moreover, Epigallocatechin-3-gallate (EGCG) facilitated osteogenic/odontogenic differentiation and mineralization of SCAPs by activating BMP2 expression and SMAD1/5/9 phosphorylation.96 Furthermore, the immune checkpoint molecule Programmed cell death 1 (PD1) that was expressed on SCAPs suppressed osteogenic differentiation by activating Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) which inhibited its downstream NF-κB signaling pathway.97 Protein arginine methyltransferase 6 (PRMT6), a member of type-I enzymes, inhibited osteogenic/odontogenic differentiation of SCAPs by downregulating C-X-C motif chemokine ligand 12 (CXCL12) expression through increasing H3R2 methylation and forming a complex with Lamin A/C (LMNA) inhibiting its nuclear entry.98In addition to the previously delineated signaling pathways orchestrating the regulatory landscape of SCAPs, an array of noteworthy molecular factors also contributes to its behavior. Researchers found HERS cells secrete SHH ligand, and it antagonized NFIC which activated Hedgehog-interacting protein (HHIP) (a Hh inhibitor) to ensure normal Hh signaling pathway activity pattern in apical papilla, promoting proper apical papilla growth and root formation.99Further insights into the regulatory network revealed that immune cells make up 83% of all tooth germ cells, demonstrating their regulatory responsibilities. They could directly modulate multiple processes during tooth development via secreted cytokines and acted on specific receptors, providing evidence for the novel roles of the immune system in tooth development.101 Under specific induction conditions, when DFSCs and DPSCs were transplanted in vivo and simultaneously treated with dentin matrix, both cells could differentiate into odontoblast-like cells, express dentin-related markers and facilitate the dentin-pulp as well as the cementum-periodontium complex regeneration. However, DFSCs possess greater proliferation potential, osteogenic differentiation capacity, and mineralization ability compared to DPSCs. In contrast, DPSCs exhibit stronger angiogenic and dentinogenic potential.102 DFSCs are primarily responsible for the formation of cementum, PDL, and alveolar bone, which is governed by a number of signaling pathways , down-regulates the Wnt/β-catenin signaling pathway, leading to suppressed osteoblast differentiation of DFSCs. The expression of DKK3 was decreased during osteogenic differentiation of DFSCs. Dkk3 knockdown enhanced the osteogenic potential of DFSCs, as evidenced by increased calcified nodules, and expression of osteogenic markers. Dkk3-deficient DFSCs also formed more osteoid matrix when implanted in vivo. These findings implied that DKK3 knockdown induced β-catenin accumulation in nuclear and activated canonical Wnt signaling pathway.104 Despite its role as a Wnt antagonist in other cell types, Naked cuticle homolog 2 (NKD2), an inhibitor of Wnt signaling pathway, enhanced the Wnt/β-catenin signaling pathway, thus promoted osteogenic differentiation of DFSCs.105The precise balance of Wnt and BMP signaling pathways is critical for determining DFSCs fate. Canonical Wnt signaling pathway was required for DFSCs differentiation. Researchers co-cultured HERS cells and DFSCs of rat molars and observed increased mineralized nodule formation by DFSCs. The results suggested that HERS cells triggered the Wnt/β-catenin pathway in DFSCs through tight junctions with DFSCs, which in turn secreted extracellular matrix containing vesicles that promoted osteogenic differentiation of DFSCs and matrix mineralization.106 An in vivo study showed BMP2 could induce cementoblast/osteoblast differentiation of DFSCs by intricate interaction between BMP and Wnt pathways to balance DFSCs fate. Exogenous WNT3a, a member of canonical Wnt signaling pathway, inhibited BMP2-induced differentiation, suggesting BMP-induced differentiation was suppressed by the Wnt/β-catenin signaling pathway. This was due to Wnt3a enhancing Lymphoid enhancer-binding factor 1 (LEF1), expression, which could inhibit RUNX2-dependent Ocn promoter activation. However, the β-catenin knockdown decreased BMP2-induced expression of RUNX2, ALP, and OCN, indicating endogenous Wnt/β-catenin signaling pathway was essential for BMP2 to induce DFSCs differentiation. BMP2 strongly induced Wnt inhibitor factor 1 (WIF1) expression, suggesting it initiated a negative feedback loop to control DFSCs maturation by inhibiting Wnt signaling pathway.107 Furthermore, the BMP2/PKA/β-catenin/DLX3 signaling pathway enhanced DFSCs osteogenic differentiation. BMP2 enhanced β-catenin transcriptional activity by activating PKA, which induced phosphorylation of β-catenin at Ser675. Activated β-catenin combined with LEF1 and SMAD4 to form a complex that promoted Distal-less homeobox 3 (DLX3) expression. Overexpression of Dlx3 further activated the Wnt/β-catenin signaling pathway. In contrast, WNT3a suppressed BMP2-induced osteogenic differentiation of DFSCs by suppressing PKA activity.108 However, WNT3a was reported to initiate osteogenic commitment, promote the early cementoblast/osteoblast differentiation of DFSCs due to it activating the canonical Wnt signaling pathway and inducing the expression of osteogenic regulators. However, it was insufficient to drive terminal differentiation and mineralization which might be due to WNT3a inducing the expression of WNT5a to negatively regulate WNT3a-induced ALP and OSX expression.110 Similarly, a study revealed that WNT3a protein administration caused OSX overexpression, which could be blocked by p38 MAPK inhibitors but not by ERK or JNK inhibitors, indicating p38 MAPK activation was indispensable for WNT3a-mediated OSX induction. P38 MAPK activation was not affected by Dickkopf-1 (DKK1), suggesting WNT3a activated p38 MAPK independently of the Wnt/β-catenin pathway. Additional studies demonstrated inhibition of p38 MAPK did not impact WNT3a-induced phosphorylation of GSK3β or the levels and nuclear localization of β-catenin. However, p38 MAPK inhibition impaired Wnt3a-stimulated β-catenin transcriptional activity.111 Expression of Secreted frizzled-related protein 1 (SFRP1), a Wnt signaling pathway inhibitor, was significantly more prevalent in PDL cells than in dental follicle, cementoblasts, and alveolar bone cells. Inhibiting SFRP1 raised the active Trimethylation of histone H3 lysine 4 (H3K4me3) histone marked on the promoters of the Runx2 and Sp7 genes, boosting their expression and stabilizing β-catenin levels in DFSCs-derived PDL cells, enhancing β-catenin transcriptional activity.112 A study found that Long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) combined with EZH2 inhibited the osteogenic differentiation of human DFSCs via blocking the Wnt signaling pathway. EZH2 was a histone methyltransferase that mediated Trimethylation at lysine 27 of histone H3 (H3K27me3). During osteogenic differentiation, EZH2 and H3K27me3 levels decreased while Wnt ligands and β-catenin increased in human DFSCs. Knockdown of lncRNA MEG3 or EZH2 promoted osteogenesis and Wnt pathway gene expression in human DFSCs. Chromatin immunoprecipitation showed EZH2 enrichment on Wnt gene promoters along with H3K27me3 accumulation, indicating epigenetic suppression. Inhibition of EZH2 reduced H3K27me3 occupancy on Wnt gene promoters, thus activating the Wnt/β-catenin signaling pathway to facilitate osteogenic differentiation.113WNT3a was strongly expressed in HERS and crucial for the formation of dental roots by interacting with the BMP signaling pathway. BMP2 could activate the AKT signaling pathway and induce Early growth response factor 1 (EGR1) expression during osteogenic differentiation of DFSCs. Inhibition of AKT prevented EGR1 expression and osteogenic differentiation of DFSCs.Pthrp+ progenitor cells differentiated into PDL fibroblasts, acellular cementoblasts, as well as alveolar crypt bone osteoblasts.21 Parathyroid hormone receptor-1 (PTH1R) was a receptor for PTHrP and is expressed in Osx+, Pthrp+ and Prx1+ progenitor cells. PTHrP-PTH1R autocrine signaling pathway was required to maintain their physiological cell fates. In Osx+ dental mesenchymal progenitors, PTH1R deletion resulted in defects in cellular survival, proliferation, and aberrant differentiation, which caused shorter dental roots, PDL loss, and ankylosis. The PTH1R signaling pathway likely acts through Histone deacetylase 4 (HDAC4), as the PTH1R-deficient root condition was partially recapitulated by HDAC4 deletion.22 PTH1R also likely suppressed Myocyte enhancer factor 2 C (MEF2C) and bone matrix proteins to prevent precocious differentiation of Pthrp+ progenitors.114Prx1+ cells were craniofacial mesenchymal progenitors expressing the transcription factor PRX1. When PTH1R was conditionally deleted in Prx1+ cells, it resulted in arrested incisor eruption and delayed molar eruption. PTH1R ablation caused decreased expression of osteogenic genes like Runx2 and Osx, resulting in reduced alveolar bone formation. In vitro, PTH1R deletion suppressed osteogenic differentiation of DMSCs. The balance of bone remodeling was disrupted due to decreased bone formation when PTH1R was knocked out in Prx1+ cells. Reduced alveolar bone growth failed to provide sufficient eruptive force for tooth eruption. PTH1R deletion also led to aberrant PDL development with decreased Periostin expression. Therefore, the PTH1R signaling pathway in Prx1+ progenitors was indispensable for osteogenesis, bone remodeling, and PDL function during tooth eruption.115 Chromodomain-helicase-DNA binding protein 7 (CHD7) promoted osteogenic potential of human DFSCs by increasing PTH1R expression and enhancing PTH/PTH1R signaling pathway. PTH1R also was a key mediator of CHD7 regulation of osteogenic differentiation in human DFSCs as it was the direct transcriptional target of CHD7.116During cementogenesis, Parathyroid hormone-related peptide (PTHrP) was mainly produced by cells in the dental follicle. These 117 In DFSCs, BMP9 transfection significantly elevated ALP activity, calcium deposition as well as osteogenic markers expression via activating non-canonical MAPK pathways, including increased p38 MAPK phosphorylation and decreased ERK1/2 phosphorylation.118. Interleukin-1alpha (IL-1α) bound to Interleukin-1 (IL-1) receptor on DFSCs and activated the downstream JNK and p38 MAPK pathways. Osteoclastogenesis was boosted and osteoblast differentiation was reduced when JNK and p38 MAPK were activated. These altered bone remodeling activities in dental follicle facilitate bone resorption around the erupting tooth and allow tooth eruption to proceed.119Gene expression profiling reveals activation of BMP and TGF-β pathways during early osteogenic differentiation.Runx2 mutation in Cleidocranial dysplasia (CCD) patient DFSCs impaired matrix remodeling and osteoclast induction in patient DFSCs, which may cause delayed tooth eruption.121 But cellular senescence in the dental follicles was reduced in CCD patients compared to healthy controls. Isolated DFSCs from the patient showed increased proliferation, accelerated cell cycle, and downregulated senescence-associated genes and proteins. The patient’s DFSCs had decreased ERK phosphorylation, indicating a suppressed MAPK/ERK signaling pathway. Inhibition of ERK reduced senescence in control DFSCs, while ERK activation increased senescence in patient DFSCs. Therefore, the presence of the Runx2 mutation in CCD prevents the cellular senescence process of DFSCs by blocking the ERK signaling pathway.122RUNX2 played important roles in regulating osteogenic differentiation, matrix remodeling, and osteoclast induction in DFSCs. 123 Laminin inhibited early but promoted late osteogenic differentiation in human DFSCs through integrin-α2/β1 and FAK/ERK pathway.14 Laminin also induced mineralization and cementogenic gene expression while suppressing ALP activity in DFSCs.124 Transient receptor potential melastatin 4 (TRPM4) knockdown enhanced osteogenesis but inhibited adipogenesis of DFSCs by modulating calcium signaling pathway and related gene expression, suggesting a regulatory function for TRPM4 played in the differentiation of DFSCs.125Activated Notch1 signaling pathway was required in the osteogenic differentiation of DFSCs. Activation of the NOTCH1 inhibited osteogenic differentiation via BMP2/DLX3 pathway in DFSCs.126Heterogeneity was crucial for maintaining the properties of DFSCs. Subclones showed decreased osteogenic ability compared to heterogenous DFSCs. Selecting subclones disrupted the heterogeneity of DFSCs and altered their gene expression. The TGF-β signaling pathway had a significant impact on subclones’ ability to differentiate due to its cross-talk with the Hippo, Wnt, and stemness pathways.127Strategies to modulate key developmental pathways could help stimulate innate dental repair mechanisms. For instance, local delivery of BMP ligands or agonists may promote HERS remodeling and odontoblast differentiation to treat root defects or stimulate reparative dentin formation. Modulating Wnt, Eda, PTH1R and FGF signalings could also direct dental stem cell behaviors. Further research should explore controlled, sustained delivery systems and optimize dosages for clinical translation.128 However, recreating the complex microenvironmental dynamics of the native dental stem cell niche remains challenging.129 Smart biomaterial scaffolds that mimic key cues like signaling pathway molecules and architecture could guide dental stem cell organization into dental tissues.130 Emerging bioprinting techniques allow precise spatial control over the distribution of cells, signals, and materials to mimic native tissue architecture and function.131 Future efforts should uncover developmental mechanisms to inform scaffold and bioink design for precision engineering of the dentin-pulp complex.132 Combined innovations in dental stem cells, biomaterials, and bioprinting have immense potential to transform restorative dentistry through bioinspired tooth regeneration.Tissue engineering approaches leveraging dental stem cells offer an attractive bio-inspired therapeutic avenue.133 Recent studies have developed protocols to acquire iPSCs-derived dental epithelial and DMSCs using precisely orchestrated signaling pathways molecules like FGFs, BMPs, and WNTs.134 For example, Kim et al. generated dental epithelial-like stem cells from human ESCs and iPSCs through a stepwise procedure involving embryoid body formation, induction with retinoic acid, and keratinocyte serum-free medium.135 The iPSC-derived epithelial cells exhibited odontogenic capacity when recombined with mouse dental mesenchyme. Other research groups have guided iPSCs to form cranial neural crest-like cells, the odontogenic precursor tissue, which was further induced into functional odontoblast-like cells secreting dentin matrix upon FGF and BMP stimulation.137 Advanced 3D culture systems like peptide hydrogels have also been harnessed to enable the CNC-like cells to reproduce dental pulp-like tissue with embedded odontoblasts and vascularization in vivo.136 Overall, iPSCs offer an exciting platform for personalized biological tooth restoration, but further optimization of tooth organogenesis and maturation protocols will be critical for clinical translation.Patient-specific induced pluripotent stem cells (iPSCs) is a prospective cell source that can be applied in tooth regeneration and personalized dentistry.141 Recent clinical trials have reinforced the translational promise of this approach in human subjects, promoting the regeneration of compromised dental pulp and periodontal tissues, thus enabling the repair of traumatized or avulsed teeth.142 This achievement in tooth tissue regeneration stands as a pivotal milestone in regenerative dentistry. To realize its full potential, it is imperative to optimize manufacturing processes, enhance regenerative efficacy, and ensure the stability of cell aggregates. In-depth explorations of interactions with host cells and tissues are essential to bolster biocompatibility. Further investigations into their performance in pathological microenvironments and the integration of biomaterials and bioactive molecules hold the potential for increased regenerative success.The application of dental stem cell-derived stem cell aggregates has successfully emulated the intricate developmental processes of odontogenic mesenchymal cell condensation, culminating in the regeneration of damaged dental pulp in murine models and the restoration of pulp-like structures and functions reminiscent of normal dental pulp.In recent decades, enormous advancement has been made in dental tissue engineering, but the clinical translation of whole-tooth regeneration remains unsolved. Several key obstacles need to be addressed, including recapitulating the intricate spatiotemporal signaling pathway dynamics during odontogenesis, optimizing vascularization and innervation of bioengineered teeth, and ensuring proper integration with surrounding hard and soft tissues. Continued interdisciplinary research on innovative biomaterials, dental stem cells, growth factors, and advanced manufacturing techniques like bioprinting will be critical to unlock the full potential of biological tooth regeneration. Seamless integration of these emerging tools and technologies will usher in a new era of next-generation, bioinspired dental treatments that far surpass traditional prosthetic tooth replacements.Elucidating the intricate developmental mechanisms directing tooth root morphogenesis is critical to advance biological tooth restoration. Spatiotemporal orchestration of signaling pathways precisely regulates dental epithelial and mesenchymal stem cell behaviors during root formation. Thoroughly decoding these complex regulatory networks promises to uncover novel therapeutic targets to incite endogenous tooth repair mechanisms. Emerging tissue engineering that based on dental stem cells has enabled the engineering of biological tooth root replacements. However, recreating the native stem cell niche environments with proper biomaterials, growth factors, and architectural cues remains a key challenge for clinical translation. Advances in these areas that accurately recapitulate developmental dynamics will be instrumental to guide organized differentiation and functional integration of dental stem cells into bioengineered root structures. Moving forward, seamless integration of interdisciplinary innovations in developmental biology, stem cells, biomaterials, and advanced manufacturing techniques is imperative to realize the immense promise of transforming regenerative technologies into biologically based tooth root regeneration therapies that restore native physiological structure and function.


Medullary thyroid cancer (MTC) is a rare malignancy, and the treatment of metastatic MTC is challenging. In previous work, immune profiling (RNA-Seq) of MTC identified CD276 as a potential target for immunotherapy. CD276 expression was 3-fold higher in MTC cells than in normal tissues. Paraffin blocks from patients with MTC were analyzed by immunohistochemistry to confirm the results of RNA-Seq. Serial sections were incubated with anti-CD276 antibody, and scored according to staining intensity and the percentage of immunoreactive cells. The results showed that CD276 expression was higher in MTC tissues than in controls. A lower percentage of immunoreactive cells correlated with the absence of lateral node metastasis, lower levels of calcitonin after surgery, no additional treatments, and remission. There were statistically significant associations of intensity of immunostaining and percentage of CD276 immunoreactive cells with clinical factors and the course of the disease. These results suggest that targeting this immune checkpoint molecule CD276 could be a promising strategy for the treatment of MTC. Medullary thyroid cancer (MTC) is the third most common thyroid cancer, accounting for 3–5% of all thyroid cancer cases . MTC oriIdentifying new treatments with minimal side effects is important. In recent work from our group, immune profiling of MTC tumors identified CD276 as a potential target for immunotherapy . CD276 iPatient demographics and the clinicopathological features of the 46 cases are presented in An excellent response to primary therapy was observed in 24 patients (52.2%). Twenty patients (43.5%) showed a biochemical incomplete response to initial therapy, whereas two patients (4.3%) showed a structural incomplete response. Twelve patients (26.1%) required additional therapy during the course of the disease. In addition to the EBRT in 12 patients, four required surgical re-treatment (8.7%). One of the 12 patients required additional use of selpercatinib (2.2%). There were four deaths among cases with MTC, but only two cases were MTC-related . The KapStatistical analysis of the IHC results was performed by dividing the study group into two subgroups. The first subgroup included those with CD276-positive samples with either 1+ or 2+ intensity and 11–50% immunoreactive cells, whereas the second group included those with samples with 2+ or 3+ intensity and 51–100% immunoreactive cells. Thirteen samples (28.3%) had a staining intensity of 1+. Intensity of immunostaining 2+ and 3+ was observed in 24 (52.2%) and 9 (19.6%) samples, respectively. The number of patients and the percentage of CD276-positive cells were as follows: 20 (43.5%) for 11–50%, 21 (45.7%) for 51–75%, and 5 (10.9%) for 76–100% . No CD27p = 0.0255). Lateral lymph node metastasis (p = 0.0010) was more frequent in patients with 2+ or 3+ intensity of immunostaining and 51–100% of CD276 immunoreactive cells. The number of patients with increased CD276 expression postoperatively who had serum CT levels ≥ 2 was 3 times higher than the number of other patients (p = 0.0036). More than 2 times more germline mutations in the RET gene occurred among patients with 2+ or 3+ intensity of immunostaining and 51–100% of CD276 immunoreactive cells than in others (p = 0.02321). A less than excellent response to initial therapy was observed in 22 patients, and this was significantly associated with 2+ or 3+ intensity of immunostaining and 51–100% of CD276 immunoreactive cells (p = 0.0374). Furthermore, statistical analysis showed that fewer patients from the 51–100% CD276-positive group achieved disease remission than other patients (p = 0.0053), and these patients were more likely to require additional therapies (p = 0.0293). The order of variables in terms of their impact was assessed based on their odds ratio (OR), calculated in relation to outcome variables at final follow-up . OR values indicate that lateral lymph node metastasis is the most important variable in both clinical contexts  (The association between the intensity of immunostaining and the percentage of immunoreactive cells with CD276 expression and histopathologic factors of MTC is presented in ctively) . Accordictively) . No statMTC is a rare tumor, accounting for 5% of all thyroid cancers, and its prognosis is worse than that of papillary thyroid cancer. The reported 10-year disease-specific mortality rate for MTC varies from 13.5% to 38% . MTC is 212Pb α-particles targets tumor cells and the vasculature, thus, showing promising effects along with low toxicity [CD276, also known as B7 homolog 3 (B7-H3), is an immune checkpoint molecule and immunoregulatory protein ,20. In 2toxicity . Li et atoxicity . Wang ettoxicity . High extoxicity . Expresstoxicity . The higtoxicity . Clinicatoxicity .In conclusion, the results of this study indicate that CD276 affects the development and course of MTC. Due to the size of the study group and the single-center nature of the study, the results obtained may not be representative of the population, which may lead to a lack of generalizability. However, given the strong correlation between clinical factors and both the intensity of immunostaining and the percentage of CD276 immunoreactive cells, there is a need for further research, which may contribute to the design of effective treatments for patients with MTC. The study included 46 patients with MTC (42 living and four dead patients) who were selected from the thyroid cancer database at the Endocrinology Clinic of Holycross Cancer Centre, Kielce (HCC). The medical records of patients were reviewed to obtain demographic information, the histological and clinical characteristics of the cancer, treatments, responses to treatment, and clinical course. The initial response to therapy was evaluated at 3 months after surgery. Responses were classified as excellent, biochemical incomplete, or structural incomplete [2O2/Amplifier, 4 min; Optiview Amplifier Multimer, 4 min; Optiview H2O2/DAB, 8 min; Optiview Copper, 4 min). Counterstaining was performed using Ventana Hematoxylin II for 8 min, followed by bluing reagent for 4 min. Finally, slides are removed from the stainer, dehydrated, and coverslipped for microscopic examination. Positive controls included a known CD276 positive human tonsil tissue. IHC slides were scored by two independent pathologists (J.K. and A.P.). The scoring criteria for staining were based on the intensity of immunostaining and the percentage of immunoreactive cells, and the percentage of CD276-positive cells and the intensity of the staining were scored. IHC-CD276 staining expression was scored as “weak” (1+), “moderate” (2+), and “strong” (3+), and samples were divided into three groups according to the percentage of CD276-positive cells as follows: 11–50%, 51–75%, and 76–100%; <10% positive cells was considered a negative result.Serial sections of formalin fixed, paraffin-embedded tissue samples from 46 patients with MTC were stained with monoclonal anti-CD276 antibody . Briefly, staining was performed on the Ventana BenchMark XT . The staining protocol included online deparaffinization, HIER  with Ventana Cell Conditioning 1 for 32 min, and primary antibody incubation for 20 min at 31 °C. Antigen-antibody reactions were visualized using the Ventana OptiViewTM Amplification kit, followed by Ventana OptiViewTM Universal DAB Detection Kit . Categorical data were summarized by frequencies and percentages. Group comparisons were performed using the chi-square test or Fisher’s exact test for categorical variables and the Mann U-test for quantitative variables .p-value < 0.05 was considered significant. All statistical analyses were performed using R .Statistical tests were two-tailed and a Study strengthOur study had several strengths. Given the currently available treatments for advanced and metastatic forms of MTC, which in a great many cases are met with the emergence of resistance among patients, it is very important to search for new treatments that will increase the chances of progression-free survival with a concomitant improvement in patients OS. In addition, new treatments should have the lowest possible rate of side effects, so conducting research in their search should focus on highly selective molecules. The present study addresses the role of CD276 in the development and clinical course of MTC, which has so far been unknown. The results considered here are the first report in the literature on the importance of CD276 in MTC and may contribute to improving treatments for this type of cancer.Study limitationsThis study has two main limitations. The first limitation is the size of the study group. MTC is quite a rare disease, as it accounts for less than 5% of all thyroid cancers, which obviously affects the number of patients available to study. However, because this type of cancer is relatively rare, the results of this study offer new and attractive information for this group of patients. The second limitation is related to the single-center nature of the study, which may contribute to the limited generalizability of its results; however, given its novel nature, its clinical value remains high.Future directionDeeper knowledge is needed to determine the specific impact of CD276 on the development and clinical course of MTC. In addition, there is a need to increase the size of the study group. The present study included patients whose final follow-up was on 31 December 2021. Additional needed is generalize the results by performing analyses based on data from other centers, which involves conducting a multicenter study. This requires obtaining acquisition of samples from other centers and analysis of newly obtained data.Practical valueExtensive literature evidence indicates that CD276 influences tumor progression. Increased expression of CD276 in tumor tissues is of interest to researchers. In turn, the difference in CD276 expression between cancer and healthy tissue suggests that targeting CD276 may be an effective therapeutic strategy. The our data identified an immune checkpoint, which may facilitate the development of new treatments in MTC. Several clinical trials are currently underway focused on CD276, and the results obtained in this study may be the basis for their continuation in this type of cancer.ConclusionsCD276 influences the development and course of MTC. In addition, it is worth noting that CD276 plays a role in the development of many types of cancer, including breast cancer, lung cancer, ovarian cancer, brain tumors, gastric cancer, and squamous cell carcinoma), and its elevated expression correlates with a worse clinical course. Therefore, CD276 can be considered as a versatile candidate for immunotherapy, capable of being used as a molecular target in the treatment of many types of cancer.