Hedgehog (Hh) signaling is highly conserved among species and plays indispensable roles in various developmental processes. There are three Hh members in mammals; one of them, Indian hedgehog (Ihh), is expressed in prehypertrophic and hypertrophic chondrocytes during endochondral ossification. Based on mouse genetic studies, three major functions of Ihh have been proposed: (1) Regulation of chondrocyte differentiation via a negative feedback loop formed together with parathyroid hormone-related protein (PTHrP), (2) promotion of chondrocyte proliferation, and (3) specification of bone-forming osteoblasts. Gli transcription factors mediate the major aspect of Hh signaling in this context. Gli3 has dominant roles in the growth plate chondrocytes, whereas Gli1, Gli2, and Gli3 collectively mediate biological functions of Hh signaling in osteoblast specification. Recent studies have also highlighted postnatal roles of the signaling in maintenance and repair of skeletal tissues. Drosophila body plan. The name Hedgehog refers to the disorganized bristles covering the hh-null embryos, which resemble hedgehog spines. Hh is secreted from a certain population in developing tissues, travels over the tissues, and regulates the activity of cells as a morphogen, which regulates the activity of recipient cells according to its concentration. The range that Hh diffuses over varies depending on the tissues: Up to 50 μm in the imaginal disc of Drosophila and 300 μm in the limb bud of vertebrates [Hedgehog (Hh) signaling plays essential roles in various developmental process in vertebrates and insects, and is a highly conserved pathway among species. Hh was first identified as a segment polarity gene by extensive screening for genes responsible for the tebrates ,2.Mammals have three hedgehog genes: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh is involved in the L-R axis determination, the patterning of body segments, the neural tube, and limbs and morphogenesis of the hair, teeth, lungs, gut, and muscle. In particular, Shh expressed in the zone of polarizing activity in developing limbs and in the notochord and floor plate of the neural tube determines the patterning of digits and cell fates of neural progenitors, respectively. Dhh regulates spermatogenesis and the formation of the peripheral nerve sheaths. Ihh is implicated in angiogenesis and hematopoiesis as well as skeletal formation ,2.A large number of studies have revealed various and indispensable functions of Hh signaling for the development and maintenance of skeletal tissues, as several concise and comprehensive reviews recently overviewed ,4,5. ThiSkeletal elements develop through two ossification processes in mammals: Intramembranous ossification and endochondral ossification. In intramembranous ossification, mesenchymal cells condense at the region where future bone is formed. The condensed mesenchyme directly differentiates into bone-forming osteoblasts. Intramembranous bones include the frontal bone, parietal bone, facial bone, part of the temporal bone, and part of the clavicle.Endochondral ossification begins with the formation of the cartilage mold; the condensed mesenchyme initially differentiates into chondrocytes, which produce a variety of glycosaminoglycan and cartilage matrix proteins, including type II, type IX, and type XI collagens. Chondrocytes proliferate and maturate. Terminal differentiation of chondrocytes is characterized by its post-mitotic hypertrophy; the hypertrophic chondrocytes express type X collagen, matrix metalloproteinase 13 (MMP13), and vascular endothelial growth factor (VEGF) and induce calcification of their surrounding matrixes. Vascular invasion is facilitated by the aforementioned vascularization-promoting factors. Hypertrophic chondrocytes eventually undergo apoptosis. The cartilage matrixes are then absorbed by chondroclasts, which are recruited into the developing cartilage along with vascular invasion.Runx2). The Runx2-positive precursors then express Sp7 to become Runx2-Sp7 double-positive precursors. Runx2 and Sp7 are master regulators of osteoblast development, as osteoblasts are absent in mutant mice lacking either of them [Alpl), secreted phosphoprotein 1 , and bone gla protein  characterize osteoblast precursors, osteoblasts, and mature osteoblasts, respectively.The fibrous layer that surrounds the developing cartilage is called the perichondrium, and it is known as a source of osteo-chondroprogenitors. Some cells in the perichondrium are specified into the osteoblast lineage. The perichondrial cell-derived osteoblasts form the bone collar, a predecessor of cortical bones. The specified population becomes bone-forming osteoblasts through several precursors. Upon their specification, perichondrial cells initially give rise to osteoblast precursors expressing runt-related transcription factor 2  and a 25-kDa C-terminal peptide (Hh-C). The Hh-N, which represents all of the Hh signaling activity, is subjected to sequentially occurring dual lipidation: Cholesterol modification to its C-terminus via covalent bonds and a subsequent palmitoylation at its N-terminus. The palmitoylation is mediated by skinny hedgehog (Ski), a membrane-bound O-acyltransferase (MBOAT). The processing and lipidation regulate not only the biological activity of Hh-N, but also affinity for the cell membrane. The affinity controls the range of Hh-N mobility over the tissue and resulting signaling gradient. Hh-C is further digested by the proteasome. The processed Hh-N is transported to the cell surface and released from the Hh-producing cells. Dispatched (Disp), a 12-pass transmembrane protein, regulates the releasing process;  in mice .Ptch genes, Ptch1 and Ptch2, have been identified in vertebrates. Cdon (cell adhesion molecule-related/downregulated by oncogenes), Boc (brother of Cdo), and Gas1 (growth arrest specific 1) are known to act as co-receptors of Ptch, enhancing the binding affinity between Hh and Ptch. Without Hh ligands, Ptch proteins are located mainly around the primary cilia and inhibit the Smo activity. When Hh ligands bind to Ptch on the target cell, Ptch exits from the cilia; the action relieves the repressive effect of Ptch on Smo, which then shuttles through the cilia, initiating Hh signal transduction [Hh signaling transduction is illustrated in sduction .In vertebrates, Hh-responsive transcription is mediated through the zinc finger transcription factors Gli1, Gli2, and Gli3 . WithoutIhh-null mutant mice clearly demonstrated the three Hh signaling-dependent processes in endochondral ossification (The phenotypes of fication : (1) ChoPTH1R; also described as the PTH/PTHrP receptor or PPR) is expressed strongly in prehypertrophic chondrocytes and weakly in proliferating chondrocytes [Pthrp, Ppr, or both Pth and Pthrp [Pthrp or the constitutively active Ppr [Periarticular proliferating chondrocytes and the perichondrium express PTHrP , whereas parathyroid hormone 1 receptor  2 and 6. These chimeric mouse studies suggest that hypertrophic chondrocytes induce bone formation at their adjacent regions by secreting Ihh. Another important point is that BMP2 and 6 produced by hypertrophic chondrocytes may not be sufficient to induce bones at this step.Hh signaling act as a master regulator of osteoblast development during endochondral ossification. In endochondral bones, osteoblasts first emerge in a perichondrial region adjacent to pre-hypertrophic and hypertrophic chondrocytes. Mouse genetic studies demonstrate that Ihh produced by these chondrocytes acts on osteoblast progenitors to execute an osteoblast specification program in the perichondrium and the primary spongiosa. hondrium ,32. The ormation ,25. In PSmo mutant mice. Ablation of Smo from perichondrial cells caused absence of Runx2 expression and bone collar formation in the perichondrium [Smo−/−/WT chimeric mice, Smo−/− perichondrial cells, which did not differentiate into osteoblasts, expressed chondrocyte marker genes including type II collagen (Col2a1) and type X collagen (Col10a1) in the region where the bone collar is formed under physiological conditions. −/−Smo cells did not contribute to bone-forming regions in the primary spongiosa [Ihh and the ablation of Ptch1 from perichondrial cells led to acceleration of bone collar formation, accompanied by the activation of Hh signaling in the perichondrium [The requirement of direct Hh input for osteoblast specification is supported by the phenotypes of hondrium . In Smo−pongiosa . In conthondrium ,33.Runx2-positive osteoblast precursors. As Smo deletion in Sp7-positive cells showed no obvious abnormality in osteoblast development, Hh signaling is dispensable for the later phase of osteoblastogenesis [The above-described studies demonstrate that Ihh produced by pre- and hypertrophic chondrocytes is essential for osteoblastogenesis in the perichondrium and the primary spongiosa during endochondral ossification . In partogenesis . Given togenesis , Hh signGli3 on an −/−Ihh background rescued abnormalities in the proliferation and maturation of chondrocytes in −/−Ihh mice [Gli3−/−; Ihh−/− mice also showed recovery of Pthrp expression in the periarticular regions [Pthrp expression in an Ihh-PTHrP negative feedback loop. Because neither Gli1−/−, Gli2−/−, nor Gli1−/−; Gli2−/− mouse embryos demonstrated any obvious defects in the growth plate [Which Gli factors act downstream of Ihh in endochondral ossification? In regulation of growth plate chondrocytes, Ihh is likely to utilize Gli3 to exert its biological effects. Removal of −/− mice ,35. The  regions ,35. Togeth plate ,36,37,38−Ihh−/ embryos were completely rescued in −Ihh−/; −Gli3−/; C2-NGli2 embryos, in which NGli2  was exogenously expressed in Col2a1-positive cells under an −Ihh−/; Gli3−/− background. Based on this result, they proposed that the Gli2 activator and the Gli3 repressor collectively mediated all major aspects of the Ihh function in endochondral ossification [In contrast, osteoblast development is mediated by all of the Gli factors, Gli1, Gli2, and Gli3, upon Hh input. The activator function of Gli2 was initially a focus in this context. Shimoyama et al. reported that Ihh induced osteoblast differentiation in a Gli2-dependent manner; they proposed that Runx2 induction and the physical interaction between Runx2 and Gli2 underlay the Ihh-Gli2 axis . Joeng afication .Gli1−/− mice showed impairment of bone formation [Gli1−/− perichondrial cells expressed Col2a1 and Col10a1, but not Runx2 or Sp7 [Gli1−/−;Gli2−/− mice showed more severe skeletal phenotypes than either Gli1−/− or Gli2−/− mice [Gli1−/−; Gli3−/− perichondrial cells compared to that in Gli3−/− cells in vitro [We have proposed that Gli1 is also involved in Hh-mediated osteoblast specification cooperatively with Gli2 and Gli3, based on the following findings: First, ormation . Second,2 or Sp7 . Third, −/− mice . Fourth,in vitro . Lastly,in vitro , and it in vitro . We alsoin vitro .Runx2 expression is lost in the perichondrium of mice defective with Hh signaling, one might expect that Runx2 is involved in Hh-mediated osteoblast specification. However, the recovery of Runx2 expression in the Ihh−/− perichondrium did not cancel the abnormality of osteoblast differentiation in Ihh−/− mice [Given that −/− mice . Thus, RKey features that distinguish cranial skeletal components forming the face and the rostral cranial vault from others in the head, limbs, and trunk are that the former components (1) originate from the cranial neural crest cells (CNCCs) and (2) generate osteoblasts via the intramembranous ossification.Neural crest cells are multipotent and self-renewing. Notably, they have a greater capacity for differentiation than the cells from which they originate: They give rise to not only ectodermal lineages, but also mesodermal lineages. In vertebrates, the neural crest progenitors are derived from the ectodermal germ layer, arising in the neural plate border, a region between the neural plate and non-neural ectoderm that forms the future epidermis. The induction of the neural plate border, i.e., the induction of neural crest progenitors, is mediated by a dynamic interplay of patterning signaling pathways: BMP, FGF, Wnt, and Notch (extensively reviewed in ).Smo in CNCCs using the Wnt1-Cre driver resulted in extensive loss of CNCC-derived skeletal elements in mice, although the generation and migration of CNCCs were not affected and the mesoderm-derived skeletons remained intact [Fox) family transcription factors , which were selected through a transcriptional profiling of Shh mutant and WT heads, were specifically downregulated in craniofacial regions of the CNCC-specific Smo mutants, which suggested that the Fox genes were direct and major mediators of the function of Hh in craniofacial development [Smo deletion affected not only intramembranous bones, but also endochondral bones including rostral half of the basisphenoid. Thus, the involvement of Fox genes in craniofacial development may be supported by a recent paper, which demonstrates that Fox genes are necessary for Sox9-mediated induction of chondrogenic genes in zebrafish [Hh signaling plays indispensable roles in the formation of CNCC-derived cranial skeletons by acting on postmigratory CNCCs. Jeong et al. found that the removal of d intact . They fuelopment . Importaebrafish .Shh, but not Ihh, is expressed in epithelial populations in the developing face from E9.5 to E12.5; Ptch1 is expressed not only in the epithelium, but also in the mesenchyme, which contains a high density of CNCCs [Ihh mutants have mild craniofacial defects [Shh is thought to be responsible for this action, since of CNCCs , and thi defects .Ihh−/− mouse cranial skeletons and the Ihh misexpression in chick embryonic heads, Abzhanov et al. speculated that in cranial intramembranous bones, the Ihh produced by mature osteoblasts acts as a feedback-inhibitor for the transition of pre-osteoblasts to the chondrocyte-like osteoblasts, which they proposed, and mature osteoblasts [Ihh was deleted in CNCCs by a Wnt1-Cre driver line [The expressions of Shh and Ihh in murine cranial sutures have been debated (reviewed in ). Ihh haeoblasts . Amano ever line . The mutMutations in cilium-related factors caused abnormalities in cranial skeletons, in line with the fact that Hh signaling transduction takes place at the primary cilia. In particular, mutations of several intraflagellar transport proteins (IFTs), which are involved in Hh signaling transduction at primary cilia, have been implicated in craniofacial development. These IFTs include IFT144  and Kif3Ihh was deleted from Col2a1-expressing cells at the postnatal stages in mice, ectopic chondrocyte hypertrophy, decreased proliferation of chondrocytes, and a disorganized growth plate was observed [Ihh in Prrx1-expressing skeletal progenitors caused lack of the growth plate and a secondary ossification center in mice [Ppr temporally cancelled the ectopic chondrocyte hypertrophy led by the deletion of Ihh from Col2a1-expressing cells, but did not correct the decreased proliferation of chondrocytes [Ppr in postnatal mice led to the acceleration of hypertrophy, followed by premature closure of the growth plate, in association with increased chondrocyte apoptosis [Several genetic studies have highlighted the roles of Hh signaling in postnatal cartilage and the growth plate. When observed . Postnat in mice . Thus, Idrocytes . These dpoptosis . Howeverpoptosis .Ptch1+/− mice showed high bone mass with a high bone turnover phenotype in adults; both bone formation and bone resorption were accelerated in the mutant mice [PTCH1 alleles [Ptch1 deletion in Bglap-expressing mature osteoblasts also led to high bone turnover in mice [Ptch1+/− mice, the mutant mice showed fragile long bones with low bone mass [Gli1 haploinsufficiency resulted in decreased bone mass with reduced bone formation and accelerated bone resorption, and impairment of fracture healing in adult mice [Gli1+/− mice supports the positive roles of Hh signaling in osteoblast differentiation. However, it is still debated how Hh signaling regulates osteoclastogenesis. Studies so far suggest not only its indirect actions via osteoblasts, but also direct actions on osteoclast precursors [We and others have demonstrated that Hh signaling also regulates postnatal bone mass. ant mice . The mou alleles . Ptch1 d in mice . Howeverone mass ,56. Thesult mice . The ostecursors , and theIhh attenuated OA progression in a surgically induced OA model compared to the WT mice [The postnatal expression of Ihh in the articular cartilage has been implicated in osteoarthritis (OA). Ihh expression is increased in human OA cartilages, whereas the expression is at the low level in healthy cartilages . Postnat WT mice . Activat WT mice . Thus, IGli1-expressing cells present in hypertrophic chondrocytes and the chondro-osseous junction at postnatal stages in mice [Gli1-expressing cells are supposed to be Ihh-responding cells in and around the growth plate. Descendants of the Gli1-expressing cells contributed to osteoblastic cells in the periosteum, trabecular bones, and cortical bones [Recently, postnatally existing skeletal stem cell-like populations have drawn attention ,63,64. S in mice . In thisal bones . These dLastly, we recently demonstrated the positive role of Hh signaling in human osteoblastogenesis and its therapeutic potential by utilizing disease-specific induced pluripotent stem cells (iPSCs) . We focuIHH and Hh signaling components, PTCH1 and HHIP (hedgehog interacting protein), as height-associated loci [Consistent with its critical roles revealed by mouse genetic studies, disruption of normal Hh signaling is known to cause human skeletal diseases. GWAS for genetic variants influencing human height identified ted loci ,69. ThisIHH also cause brachydactyly type AI , in which the middle phalanges of all the digits are rudimentary or fused with the terminal phalanges [Ptch1, a readout of Hh signaling, in the mutants’ growth plate indicates a change in the Ihh activity gradient, probably due to decreased binding capacity of the E95K-Ihh mutant to the receptor Ptch1 [GLI3 [PTCH1. Patients with Gorlin syndrome show skeletal abnormalities, craniofacial abnormalities, large body size, and tumors, including basal cell carcinomas of the skin and cerebellar medulloblastomas [Most of other Hh-related skeletal diseases are characterized by patterning defects. Heterozygous mutations of halanges ,71,72. Mh1 [GLI3 ,74,75. Tlastomas .DHCR7 mutations perturb its ability to catalyze the conversion of 7-dehydrocholesterol (7DHC) to cholesterol; the reduced cholesterol level affects Hh signaling due to reduced Smo activities [Impaired Hh signaling is thought to underlie Smith-Lemli-Opitz syndrome , an autosomal recessive multiple congenital malformation and mental retardation syndrome. SLOS is caused by mutations of the gene encoding the cholesterol biosynthetic enzyme 7-dehydrocholesterol reductase (DHCR7) , and itstivities .GNAS, which encodes Gαs [GNAS mutation is likely to underlie heterotopic ossification in POH.Aberrant Hh signaling was recently associated with ectopic ossification. Progressive osseous heteroplasia  is known to show progressive ankylosis and growth retardation caused by ectopic ossification due to mutations in odes Gαs . Regard odes Gαs . Thus, HIhh transcription is still poorly understood. How do the enhancer-promoter interaction and distal enhancer networks regulate the Ihh transcription? Is the disruption of the network relevant to human disease? Recently, Will et al. identified five enhancer regions upstream of the Ihh gene; they have overlapping activities in vivo, and a cluster of the redundant enhancers regulates the Ihh expression in skeletal development [Ihh transcription itself and those downstream of the Hh-Gli axis. This knowledge will enable us to understand the fate specification and subsequent maturation of skeletal cells, and the potential link between disruption of the process with human diseases when combined with GWAS and/or disease-specific iPSCs.As reviewed in this paper, understanding of the Hh signaling function in skeletal development has greatly advanced in the last two decades. Hh signaling not only couples chondrogenesis with osteogenesis during endochondral ossification, but also regulates intramembranous ossification in the cranial skeleton. In addition, the molecular mechanisms underlying the function have been revealed with particular focuses on Gli transcription factors and other signaling components. However, two central questions remain to be addressed. First, the gene regulatory networks underlying these actions are unclear. What genes act downstream of the Hh-Gli axis in the skeleton? What transcription factors engage the Gli-mediated network during skeletal development? The recent advancement in next generation sequencer (NGS)-based genome-wide analyses and single-cell analysis will provide insight into these questions. Second, the regulatory landscape underlying elopment . The cor


NKX2.2, leading to downregulation of negative mTOR regulators; GSK3 and AMPK are, as members of both signaling pathways, potentially important links between Hh and mTORC1 signaling; The kinase DYRK2 regulates Hh positively and mTORC1 signaling negatively. In contrast, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. Based on crosstalk observed between cilia, Hh, and mTORC1, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Although many studies focusing on crosstalk have been carried out, contradictory observations appear and the interplay involving multiple partners is far from solved. Hedgehog (Hh) signaling and mTOR signaling, essential for embryonic development and cellular metabolism, are both coordinated by the primary cilium. Observations from cancer cells strongly indicate crosstalk between Hh and mTOR signaling. This hypothesis is supported by several studies: Evidence points to a TGFβ-mediated crosstalk; Increased PI3K/AKT/mTOR activity leads to increased Hh signaling through regulation of the GLI transcription factors; increased Hh signaling regulates mTORC1 activity positively by upregulating  Crosstalk between the Hedgehog (Hh) and Mammalian target of rapamycin complex 1 (mTORC1) signaling pathways has been hypothesized by the findings that both the Hh and mTORC1 signaling pathways are involved in the progression and metastasis of several cancers [Tsc1 and Tsc2 knockdown (KD) in animal models. Thus, Tsc1 KD in zebrafish leads to phenotype characteristics of ciliary mutants [Tsc1 or Tsc2 display a phenotype of enhanced ciliary formation [Tsc1 and a shortened ciliary phenotype in cells lacking Tsc2 [Canonical Hh signaling in vertebrates is one of the most studied signaling pathways connected to the primary cilium and it is well established that a functional primary cilium is required for Hh signaling ,3. Recen mutants  and mousormation . Our owning Tsc2 . In the following sections we will shortly introduce the primary cilium and the Hh- and mTORC1 signaling pathways, followed by a summary of suggested mechanisms leading to observed Hh/mTORC1 crosstalk. The primary cilium is an immobile, microtubule-based organelle that projects as an antenna-like structure from virtually all adherent cells in the mammalian body during cellular quiescence. It functions as a sensory organelle and as a signaling compartment, by local regulation of receptors, including the G-protein coupled transmembrane receptors (GPCRs) ,7,8. TheThe BB grows from the older of the two centrioles in quiescent cells (G0 phase). The two centrioles are present in the centrosome, which is the primary microtubule organizing center (MTOC) . MTOC coThe mechanisms underlying the trafficking of receptors and signaling molecules to and within the cilium are not well understood. The movement is partly facilitated by intraflagellar transport (IFT) ,11,12,13The Hh signaling pathway is fundamental for embryonic development and for postnatal tissue homeostasis; renewal and regeneration ,15,16. DType I signals, Smoothened (SMO)-independent signals that stem from the receptor Patched1 (PTCH) and Type II signals, that are SMO-dependent. SMO-independent activation of GLI has also been classified as non-canonical Hh signaling. Hh signaling in vertebrates, involving Hh-dependent activation of GLI transcription factors via the SMO-to-GLI route, is named canonical Hh [Non-canonical Hh often refers to signaling that does not lead to activation of Glioma-associated oncogene (GLI) transcription factors . Non-cannical Hh ,19.sonic-hedgehog (SHh),desert-hedgehog, and indian-hedghog [Vertebrate canonical Hh signaling is initiated by binding of a proteolytically processed and lipid-modified Hh ligand to its receptor, PTCH. Three different Hh-related genes are present in vertebrates, -hedghog . Hh liga-hedghog . In the GLI1 and PTCH are initiated [GLI1 is routinely used as a marker for Hh activity [The GPCRs, SMO and GPR161, regulate the activity of the GLI transcription factors, in opposite directions, by regulating the level of the second messenger cAMP in the primary cilium . In the nitiated ,34,35. EChemical agonists that directly bind to SMO, such as SMO agonist (SAG) or purmorphamine also lead, independently of Hh and PTCH, to SMO accumulation in the cilium . AntagonIn the absence of Hh signaling, suppressor of fused (SUFU) has been demonstrated to regulate GLI negatively, whereas Hh signaling relieves this inhibition, but the mechanisms are far from solved. It is possible that SUFU primarily regulates GLI negatively by sequestering GLI in the cytoplasm, and in this way hampers nuclear localization ,39. HoweThe mTORC1 complex is part of the PI3K/AKT/mTOR pathway ,45. mTORTSC1 or TSC2 causes constitutive mTOR activation and development of hypomelanotic macules [TSC1 and tuberin encoded by TSC2 , form a heterodimeric complex that inactivates mTORC1 via the small G-protein Ras homologue enriched in brain (RHEB) [TSC1 or TSC2 [Loss of either  macules . The twon (RHEB) . The mTO or TSC2 . The mut or TSC2 ,55. Acti or TSC2 , which i or TSC2 ,58, whic or TSC2 . PhosphomTORC1 is located at the lysosomes, to RAB7-positive vesicles, and this location is required for its activation . The lysThe mTORC1 signaling cascade receives inputs from various upstream cues related to the metabolic and nutritional status of the cell and translates these into regulation of cell growth. The mTORC1 complex receives input according to the energy status via AMP-activated protein kinase (AMPK), mitochondria, and lysosomes . In the The AMPK complex is sensitive to the cellular AMP/ATP and ADP/ATP ratios. AMPK is a protein complex consisting of α, β, and γ subunits. When the AMP or ADP concentrations are high, AMPK is activated by phosphorylation of protein kinases, e.g., LKB1 . The actAutophagy is upregulated by AMPK-mediated phosphorylation of ULK1, leading to increased activity. The activated ULK1 upregulates the subsequent autophagy by phosphorylation of Raptor, leading to inactivation of mTORC1 ,73. DownGLI has been found amplified and expressed in several tumor types, including rhabdomyosarcoma [GLI1, GLI2, and PTCH, respectively [osarcoma ,77,78,79osarcoma ,81,82. TIn 2010, it was found that medulloblastoma resistance towards SMO inhibitors decreased as a result of combination therapy with SMO and PI3K/AKT inhibitors , and recIn summary, it is convincing that crosstalk between Hh and mTOR signaling in cancer cells exist, but the mechanisms are far from clear.Tsc1 or Tsc2 inhibit Hh signaling [Tsc1-defective MEFs is due to a reduced amount of the Hh initiation transcription factor Gli2. Furthermore, the reduced amount of Gli2 was demonstrated to be a result of the reduced activation of the Gli2 transcription factor, Smad2/3, and the reduced Smad2/3 activity, a result of a lack of functional Tsc1 required for TGFβ-induced activation of Smad2/3 [Tsc2-defective cells. In contrast to Tsc1-defective cells, the reduced Hh signaling in Tsc2-defective cells could be corrected by treatment with the mTORC1 inhibitor, rapamycin. However, the molecular background for this is unknown. We have recently demonstrated that mutations in either ignaling . We demo Smad2/3 . HoweverThese results indicate that crosstalk between Hh and mTOR can be mediated via the TGF-beta pathway .Several studies have reported importance of the PI3K/AKT/mTOR pathway in regulating the cellular distribution of the GLI proteins. In 2006, a PI3K-dependent Akt activation was found to be essential for Hh signaling in the specification of neural fates in chick neural explants, chondrogenic differentiation of 10T1_2 cells, and GLI activation in NIH3T3 cells . GLI2 phA more recent paper showed that GLI1 and GLI2 were activated by Insulin like growth factor (IGF-1)-mediated activation of the PI3K/AKT pathway in renal cell carcinoma. IGF-1 induced both GLI1/2 mRNA and GLI-Luc reporter activity, in a non-canonical manner, that could be blocked by PI3K and GLI inhibitors, but not by the SMO inhibitor cyclopamine. Moreover, the combination of GLI and AKT inhibitors significantly suppressed renal cell carcinoma growth. However, the authors did not investigate whether these observations were mTOR-dependent . Another study provided evidence that RAS/MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells in response to SHh ligand. In melanoma, both the RAS–RAF–MEK–ERK (MAPK) pathway and the PI3K–AKT pathway are shown to be constitutively active . Here, oIn the same direction, Glaucocalyxin A, which has been documented to induce apoptosis and suppress cell proliferation in various cancers, was recently demonstrated to exhibit anticancer effect on human osteosarcoma through a pro-apoptotic mechanism. Glaucocalyxin A was shown to increase apoptosis in human osteosarcoma cells, and this effect was accompanied by a concentration-dependent decrease in the nuclear concentration of GLI1, while the cytoplasmic concentration of GLI1 was increased. Glaucocalyxin A treatment also led to reduced expression of P13K and phosphorylated AKT, indicating reduced PI3K/AKT signaling. Notably, Glaucocalyxin A induced an inhibitory effect on the PI3K/AKT signaling pathway, as the decreased GLI1 nuclear localization and apoptosis were reversed by the P13K/AKT activator, IGF-1. Based on these results, it was concluded that Glaucocalyxin A inhibits GLI1 nuclear translocation via inhibition of the P13K/AKT pathway. The authors did not investigate a possible mTOR-dependent impact of this mechanism . AKT1, AKT2 overexpression was reported to regulate GLI1 nuclear accumulation and transcriptional activity in the neuroblastoma cell-line, BE(2)-M17 negatively [However, in contrast to gatively . WhetherWhile the effect of mTOR was unknown or shown not to be responsible in the studies described above, mTOR-dependent regulation of GLI, through both S6K and 4E-BP1, has been demonstrated. In 2012, it was demonstrated that S6K1 phosphorylated GLI1 at p.Ser84. Through a co-immunoprecipitation experiment, the authors found that the interaction between GLI1 and SUFU was reduced in cells with the phosphor-mimetic p.Ser84Glu mutation of GLI1, and it was suggested that the GLI1 p.Ser84 phosphorylation could reduce GLI1 binding to SUFU and in this way be responsible for nuclear translocation of GLI1. The GLI1 phosphorylation at p.Ser84 was observed upon TNF-α treatment and was suggested to increase GLI1 oncogenic function in the development of esophageal adenocarcinoma, since cells with the GLI1 mutation, p.Ser84Glu had a higher level of colony formation in a soft-agar assay. Additionally, the described regulation of GLI1 activity was SMO-independent .S6K1 did not decrease the protein level of GLI1 and overexpression of GLI1 could not rescue a reduced proliferation observed upon S6K1 KD [In contrast, a later study demonstrated that the impact of S6K1 on neuroblastoma cells was not mediated through GLI1, as KD of  S6K1 KD . This inGLI1 mRNA induction in response to SAG, and KD of 4E-BP1, by shRNAs, reversed the effect of Torin1 on GLI1 mRNA expression. Inhibition of mTORC1 further suppressed Hh-dependent growth of cerebellar and medulloblastoma [4E-BP1-mediated regulation of GLI1 has recently been demonstrated in cerebellar and medulloblastoma growth in mice. It was found that canonical Hh signaling required mTORC1-mediated inhibition of 4E-BP1, since the mTOR inhibitor Torin1 suppressed blastoma . It is iSHh. Inhibition of PI3K/mTOR by NVP-BEZ235 abrogated the SHh-induced skeletal muscle protection, indicating a protective role of mTOR-dependent Hh signaling in I/R injury [Skeletal muscles have high metabolic activity and reperfusion injury after e.g., ischemia or the use of a surgical tourniquet, to obtain a blood-free area, and may therefore cause damage and necrosis of skeletal muscles. The PI3K/AKT pathway is known to have a protective role against limb ischemia–reperfusion (I/R) injury ,97,98,99R injury .GLI genes, the authors found that nuclear localization and transcriptional activity of GLI3, but not of GLI1 nor GLI2, in Human melanoma , glioblastoma-astrocytoma (U373MG), and cervix carcinoma (HeLa) cells were regulated by rapamycin and protein phosphatase 2A (PP2A). PP2A is a protein phosphatase that acts in opposition to mTORC1 on the regulation of S6K and 4E-BP1. Inhibition of mTORC1 results in increased PP2A activity. An increase in PP2A activity led to cytosolic retention of GLI3 and reduced transcription of the GLI3 target gene, Cyclin D1, whereas reduced PP2A activity led to increased GLI3 nuclear localization and transcription of Cyclin D1. This cytosolic retention of GLI3 by rapamycin was dependent on a rapamycin-mediated activation of PP2A [In contrast, in another recent study, performed with overexpression of the  of PP2A .Together, these reports demonstrate that PI3K/AKT/mTOR can positively regulate Hh signaling, by increasing the activity of GLI1 and GLI2; also, the activity of GLI3 may be increased in case of decreased PP2A activity (and increased mTORC1 activity) . The invPTCH and SUFU resulted in increased PI3K signaling and increased phosphorylation of mTORC1 and 4E-BP1. Interestingly, decreased expression of several negative regulators of mTORC1 , leading to activation of mTORC1 was observed in human neural stem cells treated with SHh. When activated, GLI translocates to the nucleus and activates the expression of LYF and NKX2.2. The 5-kb upstream region of the negative regulators of mTOR (except STRADB) all contain transcription factor binding sites for LYF1-1 and NKX2.2. Hence, the authors suggested a model where increased NKX2.2 activity, mediated by GLI, decreases the expression of negative regulators of mTORC1 [A recent study reported a novel mechanism for crosstalk between SHh and PI3K/AKT/mTOR. The study identified Hh pathway mutations and structural copy number variants in patients with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay. These phenotypes resemble those of patients with PTEN mutations, suggesting that crosstalk between Hh signaling and mTORC1 could be involved in pathogenesis of the disease. Hh activating mutations of f mTORC1 .AKT genes, where AKT1 was reported to be a direct transcriptional target of GLI1, with two putative binding sites for GLI1 in the AKT1 promoter region [In addition to this, it has previously been demonstrated that Hh signaling modulates the expression of r region .Together, these results demonstrate that canonical Hh signaling can regulate mTORC1 activity positively, by downregulation of negative and upregulation of positive mTORC1 regulators. In mammals, GSK3 refers to the two paralogous proteins GSK3α and GSK3β, which are constitutively active serine/threonine protein kinases, involved in various cellular signaling, including Hh and mTOR signaling. The two kinases have specific substrates thus they cannot compensate for the loss of each other. However, due to the similarity of the two proteins, several chemical inhibitors of GSK3 target both GSK3α and GSK3β; thus, several studies do not discriminate between the two paralogs. GSK3β is inhibited via phosphorylation at p.Ser21 and activated via phosphorylation at p.Tyr279 ,107. TheAs described above, GSK3β is involved in negative regulation of both Hh signaling and mTORC1 activity . GSK3β hAdditional interactions of GSK3β with mTORC1 signaling, in different directions, have been reported. S6K2 and S6K1 were shown to inhibit GSK3β through p.Ser9 phosphorylation ,109. TheRecently, active mTORC1 has been suggested to be responsible for the cytosolic localization of GSK3β, as mTORC1 inhibition was demonstrated to change the cellular distribution of GSK3β from the cytosol to the nucleus, leading to phosphorylation and degradation of the nuclear located GSK3β substrate, cMyc . GSK3β pSince GSK3 regulates both Hh and mTORC1 signaling, GSK3 could be an important link of crosstalk between these pathways. However, this potential crosstalk could rely on the cellular localizations of GSK3 and the effectors of the Hh and mTOR pathways, since several components of Hh signaling localize to primary cilia and mTOR signaling is regulated at the lysosomes. In accordance with this, mTORC1 has the ability to regulate GSK3 localization (nuclear/cytosol distribution) . These sDYRK1A increased phosphorylation and activity of both TSC1 and TSC2. In agreement with this, increased phosphorylation of S6K1 and 4E-BP1 was observed in DYRK1A KD cancer cells and this effect was inhibited by rapamycin [Several examples of crosstalk between the Hh and mTOR signaling pathways that emerged through the Dual-specificity and Tyrosine(Y)-regulated kinase (DYRK) family have been demonstrated. The DYRK protein family consists of five members: DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4 ,120,121.apamycin .DYRK1B eliminated SAG-induced GLI1 induction [Dyrk1b in a MEF cell line, stably expressing SHh ligand, resulted in upregulation of the Hh target genes with increased expression of Gli1 mRNA [Dyrk1b overexpression. This increase in Gli1 protein was a result of increased Gli1 protein stability trough a mechanism that involved Akt activity, since inhibition of Akt reduced the level of Gli1 protein  [Dyrk1b transfected cells [Both negative and positive regulation of canonical Hh signaling have also been described for DYRK1B ,119. DYRnduction . The samli1 mRNA . Surpris effect) . An actied cells . Dyrk1b in NIH3T3 cells resulted in an increased mTORC1 pathway activation, verified by phosphorylation of mTor at p.Ser2448, of Akt at p.Ser473 and p.Thr308, of S6k at p.Thr389, and of S6 at p.Ser235/236. Since mTORC2 phosphorylates Akt at p.Ser473 and S6K/S6 are downstream targets of mTORC1 signaling, these results indicate a possible role of both mTORC1 and mTORC2 signaling in Dyrk1b-mediated regulation of Hh signaling [The overexpression of ignaling .Dyrk2 knockout mice (−/−Dyrk2) were generated [−/−Dyrk2 mice died at or close to birth, and multiple defects were observed, including defects in skeletal development. Investigation of the MEF cells revealed that Dyrk2 positively regulates Hh signaling as SAG-induced expression of GLI1 was significantly decreased in −/−Dyrk2. The protein level of Gli2, and to some extent Gli3, was also reduced in SAG stimulated −/−Dyrk2 MEFs, indicating that Dyrk2 is also important for the stability of the Gli2/3 proteins [In a recent study, enerated . The homproteins . Dyrk2 hproteins . It is pproteins .−/− MEF cells revealed that also the mTORC1 activity was increased [−/−Dyrk2 MEFs. Rapamycin did not affect the ciliary length [DYRK2 KD breast cancer cells. The effect of DYRK2 on mTORC1 was suggested to be through a mechanism of DYRK2-mediated phosphorylation of mTOR at p.Thr631, followed by degradation of mTOR through the ubiquitin proteasome pathway [DYRK2 in patients with hormone receptor-positive metastatic breast cancer. Patients with low expression of DYRK2 (and high mTOR activity) had a higher clinical benefit than those with high expression of DYRK2 [Investigation of the Dyrk2ncreased . Furthery length . DYRK2 h pathway . It was of DYRK2 .Thus, whereas DYRK2 regulates Hh signaling positively, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B. In contrast DYRK2 regulates mTORC1 negatively whereas both DYRK1A and DYRK1B regulate mTORC1 signaling positively.Serum starvation inhibits mTOR activity, and autophagy is induced . Both thIn 2013, autophagy was reported to regulate ciliogenesis. Upon serum starvation, the autophagic degradation of Oral–Facial–Digital Syndrome type 1 at the centriolar satellites, promoted primary ciliogenesis in MEFs . AnotherDrosophila. They showed that Hh signaling inhibits autophagosome synthesis both in basal and autophagy-induced conditions . This effect was dependent on Gli2, but insensitive to rapamycin [Contradictory observations have been published according to the interplay between Hh, autophagy, and mTORC1. A study discovered Hh to regulate autophagy in both mammals (HeLa and MEF cells) and apamycin . In contapamycin . These oapamycin . In 2015apamycin . Very recently, the IFT system was coupled with mTOR-dependent biogenesis of lysosomes in Jurkat cells. The authors reported that the IFT-B component IFT20 inhibits lysosomal biogenesis and function. Enhancement of lysosome biogenesis, observed in IFT20-deficient cells, was associated with upregulation of Transcription factor EB (TFEB) and the TFEB-dependent expression of the lysosomal gene network, in addition to reduced activity of mTOR and ERK1/2 . TFEB-deWDR35 overexpression decreased phosphorylation of S6 in a RagA-, RagB-, and RagC-dependent manner. However, the interaction between RagA and WDR35 seemed to be located at the ER and not at the lysosomes. Thus, this possible dual regulation might be mediated through subcellular translocation of proteins involved in both Hh and mTORC1 signaling, in a manner mediated by RagA and WDR35 interactions [In addition, lysosomal mTORC1 is regulated by the Rag proteins ,62. It wractions . Together these studies demonstrate that an interaction between Hh signaling, autophagy, ciliogenesis, mTORC1, and lysosomal biogenesis exists. NKX2.2, which is assumed to downregulate the negative regulators of mTOR , leading to activation of mTORC1. GSK3 and AMPK are members of both signaling pathways, and potentially important links. Both AMPK and GSK3 can inhibit Hh by phosphorylation of GLI1, promoting its degradation, and inhibit mTORC1 activity by activating phosphorylation of TSC2. The kinase DYRK2 positively regulates Hh signaling and negatively regulates mTORC1 signaling. In contrast, both positive and negative regulation of Hh signaling have been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. According to observed crosstalk between cilia, Hh and mTORC1, involving also regulation of cellular localization of the involved proteins, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Several contradictory observations have been published, indicating the complexity of this crosstalk. Different cell lines with different genetic mutations have been used, which might explain at least part of the divergence. Further research using more standardized systems is necessary.In summary, it is convincing that crosstalk between Hh and mTORC1 signaling exists. Observations from cancer cells strongly indicate crosstalk between Hh signaling and mTORC1. This assumption is supported by several studies. The mechanism behind the observed crosstalk might depend on the specific cell lines used. In Tsc1 defective cells the crosstalk between Hh and mTORC1 is mediated via the TGF-β pathway. Several reports demonstrated a mutual positive regulation between Hh and mTORC1. Increased PI3K/AKT/mTOR activity leads to increased Hh signaling, by increasing the activity of the Hh transcription factors GLI1/2/3. In similarity, increased Hh signaling positively regulates mTORC1 activity, by upregulation of


After decades of fertility postponement, we investigate recent changes in late parenthood across low‐fertility countries in the light of observations from the past. We use long series of age‐specific fertility rates from the Human Fertility Database (1950–2016) for women, and new data covering the period 1990–2016 for men. In 1950, the contribution of births at age 40 and over to female fertility rates ranged from 2.5 to 9 percent, but then fell sharply until the 1980s. From the 1990s, however, the prevalence of late first births increased rapidly, especially so in countries where it was initially lowest. This has produced a late fertility rebound in the last two decades, occurring much faster for women than for men. Comparisons between recent and past extremely late (age 48+) fertility levels confirm that people are now challenging the natural fertility barriers, particularly for a first child. Age at childbearing continues to rise across the low‐fertility countries. In 2016, mean age at first birth was often reaching 28–30 years for women, with Italy having the highest at 31 years , women's increased labor market participation, their rising levels of education, and the availability of effective contraception led to a general postponement of family formation and first births  has been attributed to a wealth of causes partly embedded in the SDT, such as efficient contraception; rising preference for competing activities such as leisure or work; greater union instability; worsening economic security, etc.  reached the levels observed in the other countries. However, the rise may have been faster as these Eastern countries lived through uncertain economic times and their family‐friendly policies were discontinued in the 1990s as policies became increasingly oriented toward the market (Sobotka 2011): Their age at first birth has increased more rapidly than in most countries of Western Europe. In addition, we expect the prevalence of late first childbirth to have increased the most in countries with severe obstacles to earlier childbearing, particularly in southern Europe and Japan. Finally, the extent of fertility postponement has increased the number of people trying to have a child at unusual ages for a first birth , and that are also distributed across the main low‐fertility areas: respectively, the German‐speaking countries, Central Europe, Southern Europe, Western Europe, and the English‐speaking countries. Japan is free standing because low fertility there preceded the other East Asian countries by 20 years  in the same way. This is done for all years across all countries. In addition, we present the sum of first, second, and all birth rates at age 48 and above in the countries where this is possible (the choice of age thresholds is explained in the introduction). To examine more closely the speed of diffusion of late births across different countries, we represent the relative increase in the contribution of first births at age 40 and above to the TFR1, taking as starting point the year of onset of postponement. Last, to compare men and women, we show the contribution to the TFR of all births at aged 40+/45+ for women, and at aged 45+/55+ for men. Contributions are shown for two time points (1990 and 2014), and we calculate the change over time separately for men and women to see whether the recent increase in the share of late births is stronger among men or women.Figure As noted before, family size limitation is a factor behind the initial decrease in late fertility, but it does not explain its subsequent resurgence. Fertility postponement, on the other hand, has increased the share of first and second births occurring after age 40. Taking the extreme example of the Netherlands, while 90 percent of births at age 40 and above in the 1950s were third or higher order births, today, 60 percent are first and second births . The contribution of first births at age 40 and above to first birth rates started increasing substantially from the mid‐1980s onwards, ranging between 2 and 5 percent in the mid‐2010s in the countries under study Figure . Late fiSecond births should also be mentioned here. The ideal number of children is predominantly two in Europe , and there are no outliers Figure , the mecThe increase in late childbearing also concerns men, among whom births at age 45+ represented up to 3.1 percent of the TFR in 1990 and up to 5 percent in 2014  were more frequent than extremely late first births. Today, extremely late childbearing mostly concerns first births. Childless women who want at least one child are challenging the natural fertility barriers, while older mothers often appear to forego a second birth.We observed that the rise in late childbearing is continuing and even accelerating in several countries. In terms of the contribution of births at age 40 and above to the female TFR, the levels of the 1950s have often not been surpassed. However, the contributions of late first births to first birth rates have risen to values not observed in the second half of the twentieth century, and certainly never before that. Today, late first births are also frequent in Central and Eastern Europe, and their prevalence across the low‐fertility countries depends largely on the time since onset of childbearing postponement in each country. Still, in countries where late first births were initially rare, the increase has been generally faster than in other countries of the same region; in the Czech Republic, Slovakia, and Estonia, it began almost immediately after the onset of postponement and was particularly fast. In Austria, postponement onset was rather late but the pace of increase in late first births was fast relative to the other Western countries.In Italy, Spain, and Japan, late first births have risen to particularly high levels, in relative and absolute terms. These countries, with very low TFRs and high levels of childlessness, offer little institutional support to combine a career and parenting and, for many years, offered a rather traditional “marriage package” to women that was unable to satisfy their general aspirations to gender equality (De Rose and Racioppi The upturn in late fertility has spawned a growing number of studies that investigate its epidemiological, social, and demographic consequences. Very good overviews of the positive and negative individual consequences of delayed motherhood in today's society are available in the literature (Mills et al. Men's reproductive lifespan extends over many more years than women's, and only after age 55 does their contribution to the TFR become comparable to that of women aged 45 or more. Though more rarely mentioned, late fatherhood also has positive and negative consequences. The positive socioeconomic consequences for their children are by and large the same as those offered by older mothers (Powell et al. The stronger acceleration of late fertility among women than men also reflects the more radical change in women's situation in recent decades. Women's life trajectories are increasingly similar to those of men (Lesnard et al. Since the upturn in late motherhood in the 1980s–1990s, particularly for first and second births, the trend has often been viewed as a “social problem,” not just by the media but also by governments (Moguérou et al. The current context of ongoing increase in late childbearing calls for reflection on ways to improve support for those who have reached later ages without being able to form a family. In some countries, this may involve a change in the legal and medical approach to ART Vialle . In addiFurther investigations are needed to assess empirically whether an age limit to childbearing, leading to a visible reduction in fertility levels, has been reached due to fertility postponement. In the mid‐2000s, Billari et al.  did not Knowledge of changes over time in fertility intentions at later ages would improve our understanding of the “demand” side of late childbearing. In Austria, fertility intentions at very late ages have risen, and the mismatch between cohort intentions to have children at later ages and late childbearing is increasing (Beaujouan Another avenue concerns the use of ART for late conceptions in different countries, an area of research so far held back by the limited availability and comparability of data (Calhaz‐Jorge et al. Supporting InformationClick here for additional data file.


Genetic factors determine how local and systemic nitrogen signals shape root system architecture in higher plants. Among all essential mineral elements, nitrogen (N) is required in the largest amounts and thus is often a limiting factor for plant growth. N is taken up by plant roots in the form of water-soluble nitrate, ammonium, and, depending on abundance, low-molecular weight organic N. In soils, the availability and composition of these N forms can vary over space and time, which exposes roots to various local N signals that regulate root system architecture in combination with systemic signals reflecting the N nutritional status of the shoot. Uncovering the molecular mechanisms underlying N-dependent signaling provides great potential to optimize root system architecture for the sake of higher N uptake efficiency in crop breeding. In this review, we summarize prominent signaling mechanisms and their underlying molecular players that derive from external N forms or the internal N nutritional status and modulate root development including root hair formation and gravitropism. We also compare the current state of knowledge of these pathways between Arabidopsis and graminaceous plant species. Plant roots preferentially take up the inorganic N forms NOpeptides . The avapeptides . ConsideArabidopsis thaliana, the root system is composed of an embryonically formed primary root and individual post-embryonic lateral roots  and small peptides. In particular, trans-zeatin has been suggested to relay a long-distance systemic N demand signal that redirects lateral root growth from low-NO3– to high-NO3– zones (3– provision stimulates isopentenyl transferase (IPT)-dependent trans-zeatin accumulation in the root and ABCG14-mediated root to shoot translocation  signatures , four of which  are expressed in roots and are targets of the monocot-specific miRNA miRNA444a  . Interestingly, reconstituted expression of either AMT1;3 or AMT1;1 in qko showed that only AMT1;3 could restore lateral root proliferation, although both transporters share similar cell type-specific localization and NH4+ transport properties  inhibit root elongation  to retain their nuclear localization, which promotes progression and emergence of lateral root primordia (3– (≥10 mM) modulates phytohormone homeostasis and/or signaling to down-regulate most, if not all, developmental processes of lateral root formation  interferes with this developmental process by inducing a systemic signal to repress the elongation of lateral roots (3– provision (30 mM) has been shown to stimulate the release of ABA from a conjugated form (ABA-glucose) by the induction of a β-glucosidase (BG1), thereby eliciting downstream an ABA response that represses lateral root growth (3– (10 mM) rapidly induces ethylene production and represses lateral root growth. Lateral root growth in etr1-3 and ein2-1 mutants, both impaired in ethylene signaling, is insensitive to high NO3–, suggesting a regulatory role for ethylene in the systemic repression of lateral root growth by high NO3–  in shoots, while RNA silencing of AtGRXS3/4/5/8 reverses the inhibitory effect of elevated NO3– on primary root growth  inhibits cell elongation, which coincides with decreased auxin levels in apical root segments  or in signaling (axr1 and axr2) are more resistant to NH4+ . The lack of HSN1 and subsequent N-glycosylation of proteins has been associated with root hypersensitivity to NH4+ (4+ triggers the production of reactive oxygen species (ROS) that act as signaling molecules in primary root elongation  was also shown to be hypersensitive to NH4+, while the detailed mechanism is unknown  of external l-Glu can remarkably inhibit primary root elongation and stimulate root branching, consequently resulting in a shorter but more branched root system  in the primary root apex, while in auxin mutants , the sensitivity of primary root growth to l-Glu is altered  signaling kinase MEKK1  in pericycle cells, the receptor protein CLV1 localizes to phloem companion cells  signaling peptides and their receptor protein, the LRR receptor-like kinase CLAVATA1 (CLV1) b, . While eon cells . Low nitTAR2. The corresponding protein catalyzes the first step in the main auxin biosynthesis route by converting tryptophan to indole-3-pyruvic acid  as being associated with a QTL of primary root length and promoting cell elongation in response to mild N deficiency. Interestingly, allelic variation caused by a single amino acid substitution from proline to leucine enhances plant BR sensitivity and signaling, which increases the root foraging response. Whereas N deficiency has no impact on the transcriptional regulation of BSK3, transcript levels of the BR co-receptor gene BAK1 increase under N deficiency, suggesting systemic N deficiency signals entering BR signaling in roots via BAK1  has been shown to sustain PIN3-mediated lateral auxin gradients across the lateral root cap and AUX1-facilitated basipetal auxin movement to antagonize the role of PIN2, thereby protecting root gravitropism under excess NH4+  signaling in shaping plant physiological and developmental processes (3– signalling. In this case, the GRAS family transcription factor gene HRS1 together with its paralog HHO1 have been reported to be transcriptionally induced by NO3– but post-translationally regulated by Pi starvation before coordinating an NO3–-dependent primary root response to Pi deficiency (4+ (3– can largely suppress second-order lateral root formation induced by K starvation (3– transport and/or signaling suggests crucial roles for the NO3– transceptor NTR1.1/NPF6.3 and the K channel AKT1 in this root response (3– and K signaling through phosphorylation of NTR1.1 and AKT1 to regulate lateral root formation (Although extensive studies have been carried out to elucidate how roots respond to N signals, phenotypic changes in root traits depend not only on the supplied N but also on the level of supply of other nutrients . Indeed,ANR1-like genes in rice or TAR2 in wheat (Over the past couple of years, several root responses to N have been investigated across plant species and found to be highly conserved. Significant progress has been made in elucidating the molecular and cellular basis of how roots sense environmental N signals, in particular by the discovery of phytohormones and peptides conveying local and systemic N signals in the model plant Arabidopsis (in wheat , future


Reactive oxygen species (ROS) are produced constantly inside the cells as a consequence of nutrient catabolism. The balance between ROS production and elimination allows to maintain cell redox homeostasis and biological functions, avoiding the occurrence of oxidative distress causing irreversible oxidative damages. A fundamental player in this fine balance is reduced glutathione (GSH), required for the scavenging of ROS as well as of the reactive 2-oxoaldehydes methylglyoxal (MGO). MGO is a cytotoxic compound formed constitutively as byproduct of nutrient catabolism, and in particular of glycolysis, detoxified in a GSH-dependent manner by the glyoxalase pathway consisting in glyoxalase I and glyoxalase II reactions. A physiological increase in ROS production  is promptly signaled by the decrease of cellular GSH/GSSG ratio which can induce the reversible S-glutathionylation of key proteins aimed at restoring the redox balance. An increase in MGO level also occurs under oxidative stress (OxS) conditions probably due to several events among which the decrease in GSH level and/or the bottleneck of glycolysis caused by the reversible S-glutathionylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase. In the present review, it is shown how MGO can play a role as a stress signaling molecule in response to OxeS, contributing to the coordination of cell metabolism with gene expression by the glycation of specific proteins. Moreover, it is highlighted how the products of MGO metabolism, S-D-lactoylglutathione (SLG) and D-lactate, which can be taken up and metabolized by mitochondria, could play important roles in cell response to OxS, contributing to cytosol-mitochondria crosstalk, cytosolic and mitochondrial GSH pools, energy production, and the restoration of the GSH/GSSG ratio. The role for SLG and glyoxalase II in the regulation of protein function through S-glutathionylation under OxS conditions is also discussed. Overall, the data reported here stress the need for further studies aimed at understanding what role the evolutionary-conserved MGO formation and metabolism can play in cell signaling and response to OxS conditions, the aberration of which may importantly contribute to the pathogenesis of diseases associated to elevated OxS. To maintain its function and structure, a cell requires energy. In cellular metabolism, energy-producing reactions from biomolecules are controlled by oxido-reductases, a group of enzymes that transfer electrons from donors to electron acceptors. High-energy, reduced molecules deriving from nutrient catabolism are oxidized in the mitochondrial electron transport chain during cell respiration, where oxygen is the final electron acceptor . The redGSH is a thiol buffer of the cell, utilized both as an antioxidant and a cofactor by many antioxidant enzymes . In the Here we describe the relationship between OxS and MGO formation and metabolism, focusing on the mechanism of S-glutathionylation as an important protective and signaling mechanism. The compartment-dependent importance of MGO pathway in relation to oxidative pressure and adaptive metabolic changes has been emphasized, together with the possible role of D-LAC. Additionally, the occurrence of S-glutathionylation in the intramitochondrial space and its relation to pathological events has been addressed. Finally, a strategic role for SLG in cell survival in the course of redox imbalance concomitantly with a positive signaling role for MGO in the reestablishment of cell redox balance in OxeS conditions is proposed.MGO is a physiological, though reactive, alpha-oxoaldehyde, mainly produced as a byproduct of glycolysis at the level of triose-phosphates DHAP (dihydroxy-acetonephosphate) and GAP  ,18. BeinIn mammalian cells, the primary route for MGO removal is the glyoxalase system. Indeed, the substrate of GLO1 is the hemithioacetal (MGO-GSH) formed in a spontaneous reaction between MGO and GSH. As the “in situ” activity of GLO1 seems to be directly related to cellular GSH level, it has been suggested that GLO1 might already be complexed with GSH in the catalytic site when it reacts with MGO . Interes+ ⇄ 2 GSH + NADP+). Therefore, NADPH and GSH are essential for maintaining cellular redox homeostasis and biological functions. However, an excessive level of both NADPH and GSH is also implicated in certain pathologies due to the induction of a condition of ‘reductive stress’, which is as harmful as OxS GSH uptake in the presence of DIC and OGC substrates and inhibitors [Mitochondria lack the enzymes for GSH synthesis and degradation. Since GSH is negatively charged at physiologic pH, it cannot penetrate cellular and mitochondrial membranes. The mitochondrial outer membrane (MOM) possesses both voltage dependent anion channels and translocase of the outer membrane . Cytosolhibitors . Since Dhibitors ,87, the hibitors . Moreovehibitors . All in Similarly, to the cytosol, the mitochondrial matrix and IMS show high GSH pool. It has been estimated that a GSH:GSSG ratio > 10,000:1, with GSSG present only in trace amounts, occurs in the matrix and IMS, and both compartments can rapidly restore glutathione redox potential after an oxidative challenge, suggesting that GSSG is efficaciously reduced to GSH . ThroughSLG has been investigated for many years to ascertain its role in cell metabolism. SLG accumulation, not accompanied by significant changes in the ratio of GLO1/GLO2 activity, occurs during the activation of human platelets , and neu−) are formed in the active site of GLO2, a process facilitated by the zinc-oxygen interaction [− is subsequently protonated by one molecule of water and released as GSH [− from GSH can be a prerequisite for the S-glutationylation of S-OH groups of target proteins, as reported for GST [Recently, it was shown that, beside the conversion of SLG into D-LAC, GLO2 might have an additional role in catalyzing S-glutathionylation of target proteins using its natural substrate SLG. Indeed, during the hydrolysis of SLG, D-LAC and the thiolate anion of glutathione  and actin by GLO2 in the presence of SLG has been shown in vitro . The cat2O2 exposition. It was shown that S-glutathionylation and deglutathionylation maintain actin polymerization/depolymerization dynamics. S-glutathionylation of actin at Cys374 prevented globular actin (G-actin) from polymerization while epidermal growth factor-induced deglutathionylation of G-actin increased significantly the rate of its polymerization to form filamentous actin (F-actin) [2O2–dependent S-glutathionylation, which can be mediated by GLO2 and other enzymes, promotes actin depolymerization, whereas glutaredoxin 1 activity accounts for its polymerization via deglutathionylation [2 consumption, and O2− and H+ release. Actin S-glutathionylation/deglutathionylation balance is then crucial to maintain a healthy level of ROS in the whole body, and is involved in respiratory burst [Since the role of GLO2 in protein S-glutathionylation has not been further investigated, a modest speculation on the possible implications of GLO2-mediated regulation of both cytosolic and mitochondrial protein function may be appropriate. As far as actin is concerned, GLO2 could contribute to the maintenance of the redox turnover of actin (actin-SH/actin-SSG) by promoting reversible S-glutathionylation in response to HF-actin) ,106. Thunylation . Actin pry burst  and angiry burst ,110,111.+ transfer from the cytosol into mitochondria by the malate-aspartate shuttle, thus elevating the cytoplasmic redox potential and NADH/NAD+ ratio, reducing O2 consumption by the respiratory chain and the activity of Krebs cycle [As mentioned above, GLO2 was also reported to induce the enzymatic S-glutathionylation of MDH in the presence of SLG, a modification that alters both MDH conformation and activity. Interestingly, it was found that the activity of GLO2 decreased in the presence of MDH, probably because the active site of GLO2 gets involved in the S-glutathionylation of MDH and is less available for SLG conversion into D-LAC . In eukabs cycle  (Figure The glyoxalase pathway is evolutionarily conserved and involved in the glutathione-dependent detoxification of MGO, a cytotoxic by-product of glycolysis. However, MGO formation and metabolism might play fundamental metabolic and signaling roles . Consist


Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as “alarmin” and driver of Th2-immunity. Aberrant TSLP expression is a hallmark of atopic diseases, including atopic dermatitis (AD). Mast cells (MCs) are overabundant in AD lesions and show signs of degranulation, but it remains unknown whether TSLP contributes to granule discharge. Degranulation of skin MCs proceeds via two major routes, i.e., FcεRI-dependent  and MRGPRX2-mediated . Evidence is accumulating that MRGPRX2 may be crucial in the context of skin diseases, including eczema. The current study reveals TSLP as a novel priming factor of human skin MCs. Interestingly, TSLP selectively cooperates with MRGPRX2 to support granule discharge, while it does not impact spontaneous or FcεRI-driven exocytosis. TSLP-assisted histamine liberation triggered by compound 48/80 or Substance P, two canonical MRGPRX2 agonists, was accompanied by an increase in CD107a+ cells (a MC activation marker). The latter process was less potent, however, and detectable only at the later of two time points, suggesting TSLP may prolong opening of the granules. Mechanistically, TSLP elicited phosphorylation of STAT5 and JNK in skin MCs and the reinforced degranulation critically depended on STAT5 activity, while JNK had a contributory role. Results from pharmacological inhibition were confirmed by RNA-interference, whereby silencing of STAT5 completely abolished the priming effect of TSLP on MRGPRX2-mediated degranulation. Collectively, TSLP is the first factor to favor MRGPRX2- over FcεRI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection. Acute mast cell (MC) activation leads to hypersensitivity reactions and is the root of disorders like rhinoconjunctivitis, asthma, urticaria, angioedema, food allergy, and anaphylaxis ,2. In thOnly certain MC-subtypes, especially those in the skin  express MRGPRX2 at high level ,18,19,20The allergic and pseudo-allergic pathways differ fundamentally in several aspects, including spatiotemporal patterns of granule discharge and dependence on IKK-β, the latter involved in SNAP23/STX4 complex formation, which is key to the so-called compound exocytosis observed following FcεRI aggregation, in which granules fuse together and are released as bigger, more heterogeneously shaped entities . InteresTogether with IL-33, thymic stromal lymphopoietin (TSLP) is widely recognized as a critical factor in allergic disorders, including rhinitis, asthma, food allergy, eosinophilic esophagitis, and atopic dermatitis (AD), i.e., conditions, in which MCs act as critical effector cells ,35,36. ABoth IL-33 and TSLP belong to the subgroup of epithelial-derived innate cytokines that support Th2 responses in part by re-programming of dendritic cells and support of innate lymphoid cells ,39,40,41TSLP, cutaneous MCs, FcεRI and, more recently, MRGPRX2 have all been linked to AD pathogenesis ,36,48, aThis was an unlinked anonymous study. Donor skins, which otherwise would be disposed of, were obtained from circumcisions (foreskin), with written informed consent of the patients or their legal guardians, as in previous studies from our group ,19,28,445/mL in Basal Iscove medium with 10% FCS  for around 3 weeks. SCF  (at 100 ng/mL) and IL-4 (Peprotech) (10 ng/mL) were provided twice a week.MCs were isolated from skin samples by a routine procedure, as described ,50. BrieThe HRA was performed according to a method routinely employed in our laboratory ,53. In bFor inhibitor studies in the absence of TSLP, cells were pretreated with SP600125 at 5 µM (JNK inhibitor) or Pimozide at 5 µM (STAT5 inhibitor) versus PAG-CM buffer (control) for 15 min and then stimulated with the above stimuli for 30 min.To study the involvement of selected kinases in TSLP’s potentiation of degranulation, cells were pretreated with the above inhibitors for 15 min, TSLP was added to selected vials, and finally the secretagogues (AER-37 or MRGPRX2 ligands) were added as above.® siRNA technology  [6/mL in Accell siRNA medium and treated with 1 µM STAT5-targeting siRNA (E-005169-00-0050), or JNK-targeting siRNA (E-003514-00-0050) or non-targeting siRNA as control (D-001910-10-50) for 48 h. After incubation, cells were primed with or without TSLP and degranulation was induced by MRGPRX2 ligands as above.RNA interference in MCs was performed according to an established protocol by using the AccellCO, USA) ,44,54. B5 cells/mL in PAG-CM buffer. Aliquots of 100 μL were stimulated by compound 48/80 (10 μg/mL), or SP (30 µM), or kept in buffer only. After incubation for 60 min, supernatants (SNs) were collected by centrifugation at 500× g, 4 °C for 3 min, and the pelleted MCs rapidly frozen at −80 °C. After thawing, aliquots of 50 μL of 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide  solution at 5 μM in citrate buffer (pH 4.5) were mixed with the same volume of supernatant or lysate and incubated for 60 min at 37 °C. The reaction was stopped by adding 100 mM sodium carbonate buffer (pH 10.7). Fluorescence intensity was measured at an emission wavelength of 460 nm after excitation at 355 nm. Percent β-hexosaminidase release was calculated as (fluorescence intensity SN/(fluorescence intensity SN + fluorescence intensity lysate)) × 100. Net release was calculated by subtracting spontaneous release, as in the histamine release assay above.β-hexosaminidase assays were run exactly as described ,50. BrieFlow-cytometric detection of CD107a cell surface expression was as described ,55. In bMRGPRX2 cell surface staining was performed according to established protocols ,28. In bIntracellular staining of signaling intermediates was performed exactly as described . In brieAll data were analyzed with the FlowJo 7.6.5 analysis software .Cell preparation and immunoblotting were performed as described . In briep < 0.05 was considered statistically significant.Statistical analyses were performed using PRISM 8 . Pairwise comparisons of TSLP priming versus control were conducted by Student’s t-test. One sample t-test (against 1) was used to calculate significance in normalized immunoblot data. For multi-group comparisons, RM one-way ANOVA with Sidak’s multiple comparisons test were applied. Fold changes (FC) of histamine and CD107a were calculated as net “parameter” after TSLP priming/net “parameter” without TSLP priming, whereby parameter refers to percent histamine release or the proportion of CD107a+ cells. The resulting FC (fold change) values were compared by Mann–Whitney test. So far, a positive impact of TSLP on MC cytokine production was reported ,57,58 anThe most selective MC function, i.e., degranulation with the release of prefabricated histamine and other mediators confined to these cells, has not been investigated or not found to be modulated by TSLP .In skin-derived MCs, TSLP alone had likewise no impact on histamine release, i.e., did neither degranulate MCs on its own nor inhibit spontaneous histamine liberation A. ConverCollectively, TSLP selectively promotes MRGPRX2-driven degranulation of human skin MCs.Degranulation is accompanied by transient detection of granule markers at the cell surface as a result of granule-plasma membrane fusion, whereby CD107a best distinguishes activated from non-activated MCs, which are visible as two clearly separated populations ,55 and iPrior treatment with TSLP led to slightly increased expression of CD107a following SP or c48/80 stimulation in a time-dependent fashion . The effSince the impact of TSLP priming seemed more robust at the level of histamine release compared to CD107a-positive cells, we quantified the respective effect of TSLP as a fold-change ratio and indeed found a significant difference between outcomes . This unCollectively, TSLP has a positive, yet modest impact on the percentage of responding MCs, while prolonged granule opening may be the reason for the more efficient histamine release.We recently elaborated that among several signaling cascades, TSLP selectively elicits STAT5 and JNK activation in skin MCs . This waThe focus of the current study was to dissect which of the cascades, if any, was required for TSLP-supported degranulation. Prior to examining this aspect, it was mandatory to realize whether the signaling components participate in granule discharge of skin MCs in the absence of TSLP.The STAT5-inhibitor Pimozide had no effect on either FcεRI- or MRGPRX2-triggered degranulation . ConversWe next explored whether JNK and STAT5 are implicated in TSLP’s promotion of exocytosis. Using a three-component setting , we found that this was indeed the case.In the overall view of all groups A,B, TSLP® mediated RNAi protocol [Exploiting our recently established Accellprotocol ,28,54, wprotocol , we finaWe conclude that STAT5 is absolutely essential for TSLP’s supportive effect on pseudo-allergic degranulation, while JNK has a further contributory role.MCs adjust their responsiveness to secretory stimuli in dependence of the tissue microenvironment, whereby modulating factors like cytokines can critically influence their secretory competence without acting as secretagogues on their own. In doing so, extracellular stimuli can modify the manifestation and course of MC-dependent diseases by diminishing or increasing the concentration thresholds of triggering factors like allergens or drugs ,62,63. HDegranulation can be efficiently elicited via two major routes in skin MCs, i.e., FcεRI  and MRGPRX2 . The former involves the high affinity IgE receptor on the MC surface, antigen-specific IgE bound to it, and the antigen  which cross-links FcεRI to start a cascade which culminates in exocytosis ,2,25,64.Notwithstanding, several themes have started to emerge. In particular, MRGPRX2-triggered degranulation can be negatively affected by cues typically supportive of the lineage. Specifically, the dominant MC growth factor SCF interferes with the pseudo-allergic route, while simultaneously strengthening allergic stimulation ,19. In aTSLP, mostly expressed by epithelial cells, is considered a Th2-promoting master switch implicated in Th2-dominated pathologies in the skin, lung and other organs, and is therefore a focus in allergy-related research ,70,71,72MC modulating activities on maturation markers, production of cytokines and survival have previously been ascribed to TSLP ,73,74,75The sensitivity of skin MCs towards TSLP can be linked to the abundant expression of the TSLP receptor in these cells ,42,44. TWe therefore asked whether STAT5, JNK, both or none form part of the apparatus, by which TSLP potentiates MRGPRX2-dependent degranulation. To be able to judge, we first needed to clarify whether JNK and STAT5 are involved in MRGPRX2-evoked degranulation in the absence of TSLP. STAT5 perturbation had no impact on histamine liberation whatsoever, and this applied not only to the MRGPRX2- but also to the canonical IgER-route studied in parallel for comparison . DependeJNK is a well-established event in the signaling cascade downstream of FcεRI that can be activated by multiple upstream kinases, including MKK7, PI3K or PKC ,85. So fHere we found a slight, yet significant effect of JNK perturbation on degranulation triggered by SP (as well as FcεRI), yet not by c48/80 . In factIn contrast to the standard situation, STAT5 activity was indispensable to the pro-secretory function of TSLP, which was nullified by Pimozide. JNK supported STAT5, although its inhibition had a less complete effect than that of STAT5. This was discernible in the ratio-centered view, whereby TSLP still had a supportive (yet reduced) effect under JNK inhibition, while this effect was virtually absent upon interference with STAT5. The indispensable nature of STAT5 was verified following its RNAi-mediated knockdown. In this setting TSLP was unable to increase degranulation in both independent experiments and to both stimuli (c48/80 and SP) . The impIn summary, while STAT5 is the dominant entity, joint action of STAT5 and JNK is likely required to optimally orchestrate the heightened secretory competence bestowed by TSLP. Synchronized activation of STAT5 and JNK may thus mediate different TSLP outcomes in skin MCs, including survival protection  and MRGPMRGPRX2 has moved to the center of attention not least because of the large and still increasing number of secretagogues activating the receptor, of which many can be linked to symptoms or diseases. These include injection-site hypersensitivity, red man syndrome, and anaphylactic reactions to an array of drugs  ,13,16,92As mentioned, aberrant TSLP activity is closely tied to AD, and tezepelumab (anti-TSLP antibody) is in clinical trials for this condition . Of impoThe relevance of overexpressed TSLP and deregulated MRGPRX2 activity in skin pathologies indicates that their synergy may have important ramifications for understanding and treatment of skin disorders, but also host-defenses organized by MRGPRX2 on the other end of the spectrum. In fact, evidence is accumulating that MRGPRX2/Mrgprb2 can mobilize anti-bacterial mechanisms, e.g., after exogenous activation in the context of Staphylococcus aureus induced dermonecrotic lesions  or by reIt remains to be elucidated whether newly synthesized mediators like cytokines can be elicited via MRGPRX2 in human skin MCs, i.e., whether this MC type behaves rather like cells of the leukemic LAD2 line (efficient production of cytokines with MRGPRX2 agonists) or rather like  primary MCs (cytokine production inefficient or undetectable) ,99. DiffAs of now, TSLP may be provisionally assumed to produce a hyperactive phenotype with selectivity for MRGPRX2, as has been suggested for aberrant KIT activity (due to SCF overexpression or the D816V mutation) in connection with allergen-dependent activation . In conc


The lung is one of several organs that can be affected by HTLV-1 mediated inflammation. Pulmonary inflammation associated with HTLV-1 infection involves the interstitium, airways and alveoli, resulting in several clinical entities including interstitial pneumonias, bronchiolitis and alveolitis, depending on which structures are most affected. Augmentation of the inflammatory effects of HTLV-1 infected lymphocytes by recruitment of other inflammatory cells in a positive feedback loop is likely to underlie the pathogenesis of HTLV-1 associated pulmonary disease, as has been proposed for HTLV-1 associated myelopathy. In contrast to the conclusions of early case series, HTLV-1 associated pulmonary disease can be associated with significant parenchymal damage, which may progress to bronchiectasis where this involves the airways. Based on our current understanding of HTLV-1 associated pulmonary disease, diagnostic criteria are proposed. The human T-cell leukaemia virus type 1 (HTLV-1) is an enveloped, single stranded RNA, human deltaretrovirus , and whiAlthough HTLV-1 preferentially infects CD4+ T cells in vivo, cell entry is achieved via the ubiquitous glucose transporter-1  and neurInfection with HTLV-1 results in serious health consequences in a minority of those infected . HTLV-1 HTLV-1 has long been considered to have a particular tropism for the lung , 27, a cMore recent studies suggest that the involvement of the lungs by HTLV-1 is not only relatively common, but can progress to clinically significant disease. In this review we will examine the literature with regards to the clinical and pathological entities that result from HTLV-1 mediated inflammation of the lung, which together comprise what will here be called HTLV-1 associated pulmonary disease (HAPD), and we will use this information to propose diagnostic criteria to further the study of this condition.Notwithstanding the frequency with which pulmonary involvement by HTLV-1 has been reported, few studies have attempted to define this risk. An association between HTLV-1 seropositivity and two specific disease entities, diffuse pan-bronchiolitis (DPB) and idiopathic interstitial pneumonia (IIP), was first suggested in 1986 in a hospital based study in an HTLV-1 non-endemic area of Japan . AntibodCommunity based cohort studies in Japan found an increased risk of self-reported asthma among men in Miyazaki  and of aA recent meta-analysis concluded that the quality of the epidemiological evidence that supports an association between HTLV-1 seropositivity and pulmonary disease was low . This, iThe poor health outcomes resulting from high rates of chronic lung disease among Aboriginal Australians has led researchers in that country to specifically focus on the association between HTLV-1 and pulmonary disease. In central Australia, where the adult prevalence of HTLV-1 exceeds 40% in some Aboriginal communities , AborigiAn association between HTLV-1 and bronchiectasis was first suggested in central Australia in 1993  and this5 peripheral blood mononuclear cells (PBMC) [5 peripheral blood leukocytes (PBL) and was more than 12-fold higher among Aboriginal adults with an HTLV-1 PVL ≥ 1000 copies per 105 PBL relative to uninfected controls [Risk of HAM increases exponentially when HTLV-1 PVL exceeds 1000 HTLV-1 DNA copies per 10s (PBMC)  and chrocontrols . An analcontrols . In subscontrols , a phenocontrols .An association between HTLV-1 infection and chronic pulmonary disease was again found in a recent cross sectional study that included 579 residents of remote Aboriginal communities and which assessed pulmonary disease blinded to HTLV-1 status using three separate methodologies. The odds of clinically defined chronic suppurative lung disease, moderate to severe expiratory airflow limitation and radiologically diagnosed bronchiectasis or bronchiolitis among adults with a high HTLV-1 PVL in peripheral blood were increased 7.1 , 9.8  and 14.4  times, respectively, in adjusted models . Althoug5 PBL) were more likely to die as a result of bronchiectasis than those who were HTLV-1 uninfected or had an HTLV-1 PVL < 1000 copies per 105 PBL [The clinical significance of HTLV-1 PVL for patients with respiratory disease was further demonstrated during prospective follow up of a hospital-based cohort . Aborigi 105 PBL . A high  105 PBL .In an HTLV-1 cohort at the UK national referral centre, 14 of 413 (3.4%) patients had CT evidence of bronchiectasis, which was higher than background rates for the UK population . ConsistMycobacterium tuberculosis in a recent meta-analysis [Mycobacterium avium-intracellulare complex infection and had more extensive disease than controls without HTLV-1 who were infected with this non-tuberculous mycobacterium [Whether an increased risk of LRTI contributes to the development of chronic pulmonary disease in people infected with HTLV-1 is unclear. HTLV-1 seropositivity was associated with a two-fold increased risk of infection with analysis . Japanesacterium . Althougacterium , 55 or macterium , HTLV-1 acterium  and Austacterium  were moracterium , 55 and acterium . Interpracterium , 58, andacterium . High HTacterium . Howeveracterium  and the A pulmonary lymphocytosis was first described in patients with HAM , and theChest X rays were abnormal for one of six cases in the initial Japanese report , and werThe development of high resolution computed tomography (HRCT) has enabled radiological evidence of pulmonary involvement by HTLV-1 to be studied in more detail. Numerous case reports and small case series have documented HRCT findings consistent with bronchiolitis in adults with HTLV-1 , 64–69, The most common radiological abnormalities identified in adults with HTLV-1 are therefore consistent with chronic inflammation involving the small airways and bronchiectasis. Less common radiological patterns are the interstitial pneumonias, which may be non-specific interstitial pneumonia (NSIP) , 73, usuStudies that compared the extent of radiological abnormalities for specific disease entities in patients with and without HTLV-1 have found more extensive CFA , DPB 3535 and brHistopathological studies of lung samples collected by biopsy or at autopsy are consistent with the radiological patterns reported above. Extensive lymphocyte infiltration involving the large bronchi and respiratory bronchioles , particuPeribronchiolar lymphocyte infiltration has also been reported from lung biopsies of patients with HTLV-1 who did not have HAM , 77, 78.Few large studies have reported the results of lung function tests for adults with HTLV-1, and their interpretation is generally limited by the absence of HTLV-1 uninfected control groups and the inability of conventional lung function tests to reliably detect small airway disease , which iAbnormal lung function has been reported for 13–30% of individuals with HAM. The diffusion capacity to carbon monoxide was reduced for seven of 30 Japanese adults with HAM . In a BrAmong individuals without HAM who have symptomatic pulmonary disease, lung function tests reveal obstructive deficits for individuals with HTLV-1 associated bronchiolitis , 77 whilIn the only community based study to date, moderate-severe expiratory airflow limitation was recorded for 30 of 316 adult participants who satisfactorily performed spirometry in remote central Australian Aboriginal communities . The adjThe strong association between bronchiectasis and a high HTLV-1 PVL , 45, 47,Broncho-alveolar lymphocytosis is largely comprised of activated T-cells.A lymphocytosis in BALF has been repeatedly found among patients with HAM , 81, 82,A large Japanese study compared PBMCs and BALF cells in four patient groups: (i) HTLV-1 with no HAM (n = 38) , (ii) HAM (n = 8), (iii) HTLV-1 seronegative controls with lung disease (n = 44)  and healthy volunteers (n = 7) . Activat2.HTLV-1 infected cells infiltrate the lung.As early as 1989, HTLV-1 DNA was detected in BALF cells, thereby confirming that HTLV-1 infected cells infiltrate into the lungs of patients with HAM , 85, 90.The difference between HTLV-1 PVL in respiratory secretions and peripheral blood may be even more pronounced when the HTLV-1 PVL in T-cells is compared . HTLV-1 3.Pro-inflammatory cytokine and chemokine levels correlate with lymphocyte numbers.The accumulation of HTLV-1 infected cells in the lungs is associated with an inflammatory milieu in that organ. Levels of cytokines, including IFN-γ and IL-2 , 95, andHigh concentrations of IFN-γ , and thePro-inflammatory chemokines CCL2, CCL5 and CCL20 are also produced by HTLV-1 infected lung epithelial cells in vitro , which mCryptogenic fibrosing alveolitis is the only discrete pathological entity for which the BALF chemokine profile has been studied. In this condition, BALF levels of CCL3 and CXCL10 are higher in patients with CFA who have HTLV-1 relative to those without HTLV-1 infection . Elevate4.Increased cell adhesion molecule expression may facilitate infiltration of HTLV-1 infected lymphocytes.Increased concentrations of several cell adhesion molecules, including sVCAM, sL-selectin, sP-selectin and sICAM , in sera5.Other possible mechanisms contributing to a pulmonary T cell lymphocytosis.S. stercoralis [The clonal proliferation of HTLV-1 infected lymphocytes after exposure to respiratory pathogens might also contribute to a T cell lymphocytosis in BALF with pathogenic potential . While trcoralis . Consistrcoralis , indicesrcoralis . This wo6.In vitro studies and animal models.HTLV-1 infects lung epithelial cells in vitro resulting in the expression of HTLV-1 tax and the amplification of proviral DNA . CytokinHTLV-1 associated myelopathy is thought to result from bystander injury as a result of chronic inflammation that is driven by a self-perpetuating, inflammatory feedback loop . InfiltrThe pathogenesis of HAPD is therefore likely to result from an HTLV-1 driven inflammatory process analogous to that proposed for HAM (Fig. No directly acting antiviral agents specific to HTLV-1 have been developed and current antiretroviral agents are ineffective in treating chronic HTLV-1 infection . ConsequClinical trials are essential to develop therapeutic options for patients with HAPD; however, these are complicated by the absence of criteria that define this condition. To facilitate patient selection for such trials, we therefore propose the following diagnostic criteria:Proven HTLV-1 infection by serology or molecular methods,Radiological evidence of bronchiolitis, bronchiectasis or interstitial pneumonia,The exclusion of other causes including rheumatological diseases and chronic infections with atypical pathogens, such as mycobacteria .5 mononuclear cells in deep respiratory secretions collected by either sputum induction or bronchoscopy. In resource limited settings, a positive qualitative HTLV-1 PCR test could be used to support a diagnosis of HAPD. However, the predictive value of qualitative HTLV-1 PCR is likely to be significantly lower than that of PVL, and PVL should be arranged for clinical trials.An HTLV-1 PVL exceeding 1000 copies per 10Where histopathological examination of lung biopsy samples is possible, typical features of lymphocyte infiltration into peribronchiolar or interstitial areas should be apparent.Clinical and pathological data indicate that the lung is one of several organs affected by HTLV-1 mediated inflammation . ConsistPulmonary disease has been reported in association with HTLV-1 subtypes A and C in genetically diverse populations in Japan , 29, the