Cancer is characterized as a complex disease caused by coordinated alterations of multiple signaling pathways. The Ras/RAF/MEK/ERK (MAPK) signaling is one of the best-defined pathways in cancer biology, and its hyperactivation is responsible for over 40% human cancer cases. To drive carcinogenesis, this signaling promotes cellular overgrowth by turning on proliferative genes, and simultaneously enables cells to overcome metabolic stress by inhibiting AMPK signaling, a key singular node of cellular metabolism. Recent studies have shown that AMPK signaling can also reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components, RAF/KSR family kinases, which affects not only carcinogenesis but also the outcomes of targeted cancer therapies against the MAPK signaling. In this review, we will summarize the current proceedings of how MAPK-AMPK signalings interplay with each other in cancer biology, as well as its implications in clinic cancer treatment with MAPK inhibition and AMPK modulators, and discuss the exploitation of combinatory therapies targeting both MAPK and AMPK as a novel therapeutic intervention. The Ras/RAF/MEK/ERK (MAPK) signaling is a fundamental pathway in cell biology, and its alteration causes human cancers or developmental disorders. Given its crucial roles in physiology and pathology, this pathway has been extensively studied for over two decades. Unfortunately, the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity and diverse crosstalks with other signalings. Here, we focus on the complicated interplays between the MAPK and the AMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies. We hope this review would provide a conceptual framework for developing more effective therapeutic approaches against hyperactive MAPK signaling-driven cancers.–1980s, when Ras small GTPases were identified as first oncogenes from sarcoma viruses [The Ras/RAF/MEK/ERK  signaling is a central pathway that regulates cellular proliferation, differentiation, and survival. This signaling pathway was discovered in the 1970s viruses . Later,  viruses . In cont viruses . Followi viruses , 13, resThe first component of MAPK signaling, Ras small GTPases, have three gene isoforms: H-ras, K-ras, and N-ras, that encode four proteins with splicing isoforms of K-ras giving rise to K-ras4A and K-ras4B. Although all Ras proteins possess highly homologous sequences, they have quite different activities, tissue expression patterns, and effector preferences, which lead to their differential physiological and pathological functions –17.The downstream of Ras small GTPases is the RAF/MEK/ERK kinase cascade . The firIt should be noted that active Ras also turns on other signaling pathways such as PI3K/AKT/mTORC, which regulate different cellular functions . In thisThe MAPK signaling plays a crucial role in cell biology and is tightly regulated in normal cells. Upon engagement of receptor tyrosine kinases (RTKs) or other stimulations, Ras small GTPases are activated by GTP/GDP exchange factors (GEFs), which in turn recruit RAF/MEK complexes to the plasma membrane and trigger the RAF/MEK/ERK kinase cascade through facilitating RAF/RAF (or KSR), RAF/MEK, and MEK/MEK interactions as well as subsequent phosphorylations . Active Hyperactive MAPK signaling exists in over 85% of cancers, which is caused directly by genetic alterations of its upstream activators or components, including RTKs, Ras, and BRAF, or indirectly by those independent of Ras or RAF –66, and Given their high prevalence in cancers, great efforts have been made to develop specific inhibitors against oncogenic Ras and RAF mutants in the last decades. These inhibitors that have been approved for clinic treatment of Ras/RAF-mutated cancers or under clinical trials are listed in Table It has been shown that Ras activates downstream effectors through direct interactions. Therefore, disrupting Ras/effector interactions might be an alternative approach that can effectively block cancer growth driven by Ras mutations. Such a type of small molecule blockers include rigosertib, sulindac, and MCP110, and among which, the therapeutic efficacy of rigosertib combined with nivolumab for Ras-mutated cancers is being determined by phase I/II clinical trials currently   is an energy sensor that monitors the AMP:ADP:ATP ratio in eukaryotic cells. This atypical protein kinase was firstly discovered as a contaminant during the purification of acetyl-CoA carboxylase (ACC), a well-studied substrate of AMPK for fatty acid (FA) synthesis nowadays –136 Fig. HoweverAs a key sensor of cellular energy stress, the activity of AMPK is predominantly regulated by cellular AMP/ADP/ATP that competitively binds to the γ subunit of AMPK and thus promotes or inhibits the phosphorylation of Thr172 on α subunit by the tumor suppressor liver kinase B1 (LKB1) or the dephosphorylation of this site by phosphatases , 155  that requires acetyl-CoA in its reduction reaction , 165 Fi. Also, AActive AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above. Activation of AMPK stimulates the expression and plasma membrane translocation of solute carrier family member (GLUT) proteins and thereby facilitates glucose import , 176–181Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammalian target of rapamycin complex 1 (mTORC1). The mTORC1 is a central sensor of cellular amino acids that samples amino acids in both cytosol and lysosome , 185. UpIt is well known that AMPK is a putative substrate of tumor suppressor, LKB1 , 191 Fi. TherefoG12D-driven non-small cell lung cancer, the failure of AMPK activation by virtue of LKB1 mutation sensitizes cancer cells for phenformin-induced metabolic stress, further supporting that AMPK adapts cancer cells for metabolic stress [Although significant studies have shown that AMPK dampens the pathogenesis of cancers, some emerging findings indicate that it may promote disease progression under other circumstances. In T cell acute lymphoblastic leukemia (T-ALL), oncogenic Notch signaling induces a high level of aerobic glycolysis which needs to be restrained by AMPK, and loss of AMPK results in energy stress-driven apoptosis of leukemic cells and slows down disease progression . Similarc stress . Alternac stress . Consistc stress . In addic stress . Besidesc stress . All theUnlike LKB1, which is frequently mutated or deleted in cancer genomes –210, AMPAs described above, the MAPK signaling controls cellular proliferation, differentiation, and survival, whereas the AMPK signaling regulates cellular metabolism. However, many studies have indicated that these two distinct signalings have profound and complicated interplays in both physiological and pathological processes. In quiescent cells, the AMPK signaling maintains energy homeostasis by switching on catabolic pathways that generate ATP, while switching off anabolic pathways that are required for cell growth , 212–215The interplays between MAPK and AMPK signalings are binary, and the AMPK signaling can regulate MAPK signaling reversely. Conclusive evidence shows that AMPK can directly phosphorylate the RAF/KSR family kinases, the pivotal components of MAPK module, and alter their activities under variable conditions. It is well established that the hetero-/homo-dimerization of RAF/KSR family kinases plays a determinant role in the activation of MAPK signaling, which requires the association of 14-3-3, a dimeric scaffold protein with their carboxyl-terminus , 229. MeThe interplays between MAPK and AMPK signalings also alter cellular autophagy, particularly that of cancer cells. Cancer cells with Ras/RAF mutations have much higher activity of autophagy –240, whiHyperactive MAPK signaling is responsible for a large portion of cancers, and genetic alterations that aberrantly activate this pathway mainly occur on receptor tyrosine kinases (RTKs), Ras small GTPases, and BRAF . In currIt has been shown that the components of MAPK signaling form a super complex in cancer cells with active Ras , 274, whSince most oncogenic Ras mutants are undruggable, efforts for developing effective approaches against Ras-mutated cancers have been switched to identify and target synthetic lethal vulnerabilities of Ras mutants over decades, which led to the discovery of some putative factors essential for in vitro growth of Ras-mutated cancer cells , 279. UnCombinatorial inhibition of both MAPK and AMPK signalings has shown promising potentials for treating Ras/RAF-mutated cancers. To target MAPK signaling, the first-generation RAF/MEK/ERK inhibitors have been developed and applied to clinic treatment, and the second-generation drugs that can inhibit RAF mutants with elevated dimer affinity or have much less paradoxical effect have also undergone clinical trials , 282. InRecent studies have provided compelling evidence that interplays between MAPK and AMPK signalings play a critical role in cell physiology and have important implications in disease treatment, particularly for cancer. Combinatory targeting both MAPK and AMPK signalings represents for a promising therapeutic intervention. However, although the framework by which these two signalings interact with each other has been illustrated, the precise molecular basis and their impacts on cancer therapies remain largely unresolved. For instance, how the AMPK signaling differentially regulates the dimerization of different RAF isoforms (BRAF versus CRAF) and thus distinctly alters the outputs of MAPK signaling in Ras- versus RAF-mutated cancers is unclear. Besides elevating autophagic flux, does the AMPK signaling plays other roles in the MAPKi-resistance of Ras/RAF-mutated cancers? Addressing these questions would deepen our understanding of MAPK/AMPK interplays and help us develop better combinatorial therapies for cancers and other diseases. In addition, developing AMPK-specific activators/inhibitors would be an attractive research topic for both academy and pharmaceutical industry in the next years given their absence and unmet needs in clinic treatment.


A 61‐year‐old Japanese man presented with a 2‐month history of colicky abdominal pain and watery diarrhea. He had begun organic farming using cattle manure 1 year previously. Giardia duodenalis trophozoite was detected in a fecal smear  and albumin (3.5 g/dL) without inflammation (C‐reacted protein 0.1 mg/dL). Abdominal computed tomography revealed small intestinal wall thickening , foul‐smelling fatty stools (75%), and abdominal cramps and bloating (71%).G duodenalis cysts in feces of mammals like cattle, sheep, and dogs through the fecal‐oral route. Outbreaks often occur via contaminated water or food. In Japan, about 100 cases have been reported for public health surveillance, and an outbreak occurred in 2010.G duodenalis through sewage water or feces. Transmission is often associated with farmers and veterinarians. Handwashing and glove use are important for preventing transmission.Giardiasis is an intestinal infection caused by The authors have stated explicitly that there are no conflicts of interest in connection with this article.All authors had access to the data and a role in writing the manuscript.We have obtained written informed consent of the patient for publication.Video S1Click here for additional data file.


During embryonic development in vertebrates, morphogens play an important role in cell fate determination and morphogenesis. Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-β (TGF-β) family control the dorsal–ventral (DV) patterning of embryos, whereas other morphogens such as fibroblast growth factor (FGF), Wnt family members, and retinoic acid (RA) regulate the formation of the anterior–posterior (AP) axis. Activation of morphogen signaling results in changes in the expression of target genes including transcription factors that direct cell fate along the body axes. To ensure the correct establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by a fine-tuning of morphogen signaling. In this review, we focus on the interplay of various intracellular regulatory mechanisms and discuss how communication among morphogen signaling pathways modulates body axis formation in vertebrate embryos. Drosophila embryo that contains many nuclei in a large cytoplasm [A morphogen is defined as a molecule released from a localized source that determines several different cell fates and controls morphogenesis by regulating gene expression in a concentration-dependent manner ,2. Durinytoplasm .Xenopus embryos, the regulation of body axis formation by morphogens has been thoroughly investigated, and it has been shown that a gradient of BMP signaling determines the dorsal–ventral (DV) axis  are expressed, cannot receive BMP signaling and adopt a neural fate [ap-2 (tfap2a), dlx3/5, vent-2 (ventx2.2), and msx1 on the ventral side of gastrula embryos and downregulates the expression of neural marker genes such as sox2 and ncam [In early DV) axis . During ral fate ,23,24,25ral fate ,30,31,32and ncam ,36,37,38hoxa7, hoxb9, hoxc6, and caudal-related genes, that have important roles in the patterning of the AP axis; some of these genes have also a role in suppressing anterior development [cad3 and meis3 that are important for posterior development [hoxa1, hoxb1, hoxa3, hoxd4, and vhnf1 that pattern the posterior part of the brain [The anterior-posterior (AP) patterning of embryos is regulated by FGF, Wnt, and RA signaling  8,39,4040,41,42.elopment ,45,46,47elopment ,50,51,52elopment ,54. RA ielopment ,55,56. Rhe brain ,57,58. Ihe brain ,61,62,63To ensure the correct organization of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by the fine-tuning of morphogen signaling. In the following parts of this review, we discuss how communication among morphogen signaling pathways, especially BMP, FGF, Wnt, and RA signaling, is achieved intracellularly and functions as the molecular link that coordinates DV and AP patterning during body plan formation in vertebrates.Xenopus embryogenesis, FGF8 and insulin-like growth factor 2 (IGF2) promote neural induction  by inhibition of BMP signaling via MAPK-mediated Smad1 linker phosphorylation (inhibition path (a) in Xenopus and zebrafish embryos [Several intracellular factors have been shown to function as molecular links between morphogen signaling pathways that coordinate DV and AP patterning in the embryo. A well-studied intracellular factor is Smad1, which primarily transduces BMP signaling. Smad1 has a structure consisting of three domains: Mad-homology 1 (MH1), MH2, and a linker region between the MH1 and MH2 domains ,64. The  embryos ,70, furtin vitro [gadd45 [Drosophila, it has been reported that Mad (the Drosophila homolog of Smad1) is used in both BMP and Wnt signaling pathways [The molecular mechanism linking Smad1 phosphorylation and BMP, FGF, and Wnt signaling has been identified ,71,72,73in vitro . This fi [gadd45 . Moreovepathways . While tsmad1 or smad5 die at E9.5–E11.5; however, embryos that are null for smad8 survive and develop normally [smad1+/- and smad5+/- heterozygous mice are viable and fertile, smad1+/-; smad5+/- double-mutant embryos die around E10.5 [smad1 mutant mice lacking the MAPK phosphorylation site in the linker region show defects in gastric epithelial homeostasis but otherwise show normal early development [In zebrafish, Smad1 and Smad8  have a redundant function in the DV patterning of embryos ; in the normally ,86,87. Melopment . Therefosix3 (six homeobox 3), a vertebrate homolog of the Drosophila sine oculis gene [six3 mutant mice show abnormal craniofacial morphogenesis and lack eyes, nose, and most head structures anterior to the midbrain [Xenopus, Six3 represses bmp4 expression (inhibition path (c) in chordin. Thus, Six3 maintains and refines the size of anterior neural tissue by protection against the ventralizing activity of BMPs. Although Wnt signaling is capable of inhibiting six3 expression, Six3 can repress the expression of wnt1 and wnt3 in the anterior neuroectoderm that is fated to become the forebrain during mouse and chick development (inhibition path (d) in fgf8 show expanded expression of six3 toward the posterior ectoderm and fail to form a neural tube [six3 is limited to rostral neural tissue by FGF signaling in mouse embryonic stem cell aggregates that intrinsically develop a rostral–caudal neural pattern [six3 by Wnt and FGF signaling is necessary to achieve the correct patterning of the ectoderm along the AP axis. Thus, by integrating BMP, Wnt, and FGF signaling, Six3 functions as a key molecule that regulates DV and AP patterning of the ectoderm.The gene lis gene , plays alis gene ,91,92,93midbrain ,95. In zral tube . In acco pattern . These feven-skipped homeobox 1  is expressed in the posterior regions of mouse and Xenopus embryos during gastrulation [Xenopus, Evx1 overexpression causes anterior truncation and the induction of posterior marker genes during early development [Xenopus embryos results in the reduced expression of posterior markers and a failure of trunk/tail development [evx1-null mice needs to be analyzed, conditional mutation of evx1 affects commissural axon projections in the developing spinal cord [The transcription factor rulation ,102,103.elopment ,105,106.elopment . Similarelopment . Althougnal cord . aldehyde dehydrogenase 1 family member A2/raldh2 , which synthesizes RA from its precursor [aldh1a2, Evx1 suppresses the expression of the RA-degrading enzyme cytochrome P450 26 (cyp26) to activate RA signaling and further promote posterior development [cyp26 [evx1 [sox3 and hoxb1 [Zebrafish Evx1 induces the expression of recursor . As RA arecursor ,19,56. Ielopment . FGF andt [cyp26 ,110 and 26 [evx1 ,113,114.26 [evx1 . Accordind hoxb1 . Hence, Xenopus foxb1 is detected in the posterior dorsal ectoderm of early gastrula embryos and, at later stages, in the mid- and hind-brain and spinal cord [foxb1 is induced by the posteriorizing factors FGF and Wnt. Moreover, we found that Xenopus foxb1 acts as a downstream gene of Oct25 (Pou5f3.2) that inhibits BMP responses; FoxB1 also promotes neural induction at the expense of epidermal differentiation [Xenopus ectodermal cells. Upon BMP stimulation, pSmad1/5/8 translocates into the nucleus and undergoes dephosphorylation of its C-terminal sites by protein phosphatases, followed by recycling via nucleocytoplasmic shuttling [Forkhead box B1  is a member of the forkhead box (Fox) transcription factor family and contains a characteristic DNA-binding domain with a winged helix motif ,116. Micnal cord . The expntiation . Overexphuttling ,125,126.huttling . ThroughXenopus showed that FoxB1 is required for the formation of posterior neural tissue and the suppression of anterior development [wnt8, fgf3, and fgf8); overexpression of these genes can rescue AP patterning defects in FoxB1-knockdown embryos (activation path (h) in Xenopus embryogenesis through the regulation of Wnt and FGF signaling pathways. Although the inhibition of endogenous FoxB1 function does not cause significant defects in DV patterning, the double knockdown of FoxB1 and Oct25 results in a severe reduction in the expression of the neural marker sox2 and causes the expansion of epidermal keratin  expression into neural plate territory. FoxB1 functions both downstream of and in concert with Oct25; therefore, FoxB1 forms a feed-forward network with Oct25 which is important for induction and/or maintenance of neural tissue. In summary, FoxB1 controls the establishment of the DV and AP axes of the ectoderm by modulating BMP, Wnt, and FGF signaling.Knockdown of FoxB1 in elopment . FoxB1 uXenopus Zbtb14 promotes neural tissue formation at the expense of epidermis in early embryos [Xenopus embryogenesis. Intriguingly, mice expressing a C-terminally truncated form of Zbtb14 show severe defects in heart, kidney, and brain organogenesis [zbtb14 knockout mice are needed to clarify the role of Zbtb14 in body axis formation in mammals. As dysregulation of BMP and Wnt signaling components also leads to malformations in heart, kidney, and brain [Zbtb14  is a zinc-finger and BTB/POZ  domain-containing protein ,129,130, embryos . Similar embryos ,136,137.ogenesis ,139; furnd brain ,146,147,Xenopus embryos [Xenopus clk2 is expressed in neural tissues along the AP axis during early embryogenesis [epidermal keratin is also reduced in embryos overexpressing Clk2, and this suggests that Clk2 promotes dorsalization/neural induction. Clk2 interferes with BMP signaling downstream of BMP receptor activation, and the neural-inducing ability of Clk2 is enhanced by both BMP inhibition and activation of FGF signaling. Mechanistically, Clk2 downregulates the level of pSmad1/5/8 in cooperation with BMP inhibition and increases the level of activated (diphosphorylated) MAPK induced by FGF signaling (inhibition path (g) and activation path (h) in clk2 is one of the causative genes of ASD and is therefore a potential therapeutic target. In addition, abnormal brain outgrowth has been observed in ASD patients [Xenopus, overexpression of Clk2 expands the neural plate by regulating BMP and FGF signaling during early development [We recently reported that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermal differentiation in  embryos . Clk2 is embryos ; it has  embryos ,151,152.ogenesis . Overexppatients . In Xenoelopment . Thus, tclk1 is expressed in the mammalian brain and induces neuronal differentiation of PC12 cells [clk2 gene show hepatic lipid accumulation when fed a high-fat diet [clk2-null mice has not yet been reported. In future studies, combinatorial inhibition of Clk family members is needed to clarify the role of Clk in body axis formation. The Clk family consists of four paralogs (Clk1–4). The paralog 12 cells . Moreove12 cells . Therefofat diet ; howeverIn early vertebrate embryos, BMPs determine the DV axis by inducing ventral fate, and the AP patterning is regulated by FGF, Wnt, and RA signaling pathways. In this review, we focused on the molecular links that coordinately regulate the processes of DV and AP axis formation through the fine-tuning of morphogen signaling. Recent advances have revealed an increasing number of intracellular molecules that are important for the integration and balancing of morphogen signaling pathways . Since g


Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders. St. St13]. ignaling ,15,16;  and non-canonical (kinase-independent molecular interactions) function of GRKs in GPCRs regulation has been well-described and reported in many mammalian cell types. However, the presence and function of GRKs in platelets has been a more recent area of study. Messenger RNA and predicted protein copy number analysis in human and mouse platelets have identified the presence of GRK expression ,22,23. F2+ elevation, RAP1 activation, and integrin outside-in signaling . Tx. Tx2 is lization . Notably isoform . We havelatelets ,26.12, but not P2Y1, is mediated by GRK2 and GRK6 [12 receptor desensitization. Most recently, using GRK6 deficient mouse platelets, Chaudhary et al. reported that GRK6 is critical for regulating platelet activation through PAR4- and P2Y12-selective GPCR desensitization [1-mediated signaling. Further studies are needed to validate the role of GRK6 in P2Y1-depedent signaling in platelets.Consistent with what we have documented, Hardy et al. also reported in astrocytoma cells that the desensitization of the P2Yand GRK6 , supporttization . In cont2A adrenergic receptor- and serotonin 5HT2A-mediated platelet activation [In addition, Chaudhary et al. demonstrated that GRK6 is not involved in the regulation of epinephrine αtivation . Our restivation . Taken tTermination of GPCR signaling requires not only the GRK-mediated phosphorylation of the receptors but also the function of arrestins, which are recruited and bound to activated GPCR. The activated GRCR/arrestin complex concentrates in punctate areas of the plasma membrane, where they co-localize with endocytic machinery like clathrin and AP2 ,47. In a1 and P2Y12 desensitization. Using both laser and ferric chloride injury models, Schaff et al. demonstrated that deletion of arrestin-2 in mice, but not arrestin-3, results in a decreased thrombus formation. They further revealed that arrestin-2 promotes thrombus formation through its participation in integrin αIIbβ3 signaling, suggesting a direct signaling function of arrestin during platelet activation. It is still unclear whether arrestin-2 or arrestin-3 has functional redundancy in platelets for certain GPCRs, which contributes to the observed phenotype. Another study by Li et al. showed that the contribution of arrestins to PAR-mediated signaling has been limited to PAR4 [12 heterodimerization is involved in the recruitment of arrestin-2 to PAR4, where it has a positive signaling role. These two studies both indicate a direct signaling role for arrestin-2 instead of the expected desensitization of GPCR-dependent signaling in platelets. Whether the classical role of arrestin, the facilitation of desensitization of GPCR signaling observed in many cell types, exists in platelets remains elusive.A previous study by Schaff et al. using arrestin-2 and/or arrestin-3 knockout mice showed that platelet activation is not altered in the absence of arrestin-2 and arrestin-3 . Further to PAR4 . PAR4-P22+ mobilization in response to some GPCR agonists in arrestin-3 knockout platelets, mainly downstream of PAR4- and P2Y12-mediated signaling pathways [A recent study using arrestin-3 knockout mouse platelets has for the first time identified the negative regulatory function of arrestin-3 in platelets, which is demonstrated by increased platelet aggregation, secretion, integrin activation, and Capathways . Overall12-mediated signaling  regulates desensitization of P2Y1 receptor [It has been long recognized that during platelet activation, GPCRs undergo desensitization following activation. For example, PAR1 undergoes rapid desensitization due to internalization, whereas activation-dependent internalization of PAR4 is much slower ,81,82,83truction . Subsequtruction . Baurandr injury . In contreceptor ,87. MoreInterestingly, GPCR desensitization has generally been thought of as a late signaling event and functions as a “protective mechanism” in platelets. However, this statement has not been experimentally validated in vivo. In other cell types, GPCR desensitization can occur rapidly within seconds . Collect-/- mice generated by CRISPR-Cas9, Chen et al. examined the consequences of GRK6 knockout on platelet activation and accumulation at the site of injury using the cremaster laser injury model. As discussed above, the thrombus architecture that forms consists of a core of tightly packed P-selectin positive platelets overlaid with a shell of loosely adherent P-selectin negative platelets. We found that platelets accumulate more rapidly at the site of injury in GRK6-/- mice than in WT littermates , and ADP P2Y12 signaling . Besides the above reagents, a number of PAR1 and PAR4 antagonists are currently being evaluated in clinical trials [Many antiplatelet drugs target GPCRs and their signaling pathways of platelet activation, including PAR1 antagonist (vorapaxar), thromboxane Al trials . In the l trials . TherefoThere is heritable interindividual variation in platelet reactivity that may be relevant to clinical events, such as myocardial infarction , and thiA non-synonymous SNP  in GRK5 is associated with differential survival in African American heart failure patients . In a stCurrently, the novel coronavirus has affected many people worldwide and finding a cure is a top priority. Patients with COVID-19 have been found to face increased thrombotic risk ,102,103.From a molecular point of view of the COVID-19 infection pathway, angiotensin-converting enzyme 2 (ACE2), to which the SARS-CoV-2 viral particles bind, is a critical component of the renin-angiotensin-system (RAS). ACE2 converts angiotensin II (Ang II) into Ang 1–7. These biological active fragments may subsequently activate the angiotensin II receptor type 2 (AT2). AT2 is a class of G-protein coupled receptors. Interestingly, the GRK-arrestin system negatively regulates AT2-mediated signaling ,110. ThuIn the past three decades, GRKs have been shown to play an important role in the heart by regulating GPCR signaling. Therefore, GRKs have been extensively studied as therapeutic targets in cardiovascular disease. However, there is a critical gap in knowledge between the unexplored function of GRKs in platelets and their well-studied role in cardiovascular health and diseases. The work published to date about GRK6 and arrestins in platelets may reveal only the “tip of the iceberg” of GRKs biology in hemostasis and thrombosis. Thus, ongoing studies may reveal meaningful information about 1) the role of GRKs in hemostasis and thrombosis and 2) the function of GRKs in platelets versus nucleated cell types. The insight gained from new approaches, such as genome-wide association studies (GWAS), expression quantitative trait loci (eQTL) analysis, CRISPR-Cas9 mediated genome-editing, and human iPSC cells will advance our understanding of 1) the role of GRKs in the cardiovascular system and 2) the effect of GRK genetic variants on platelet reactivity in general and in the context of racial differences. In summary, not only will these areas of investigation enable us to have a better understanding of GPCR regulation by GRKs, but they may also ultimately lead to new therapeutic options for the treatment of cardiovascular and thrombotic disorders.


Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGFβ) superfamily of cytokines. While some ligand members are potent inducers of angiogenesis, others promote vascular homeostasis. However, the precise understanding of the molecular mechanisms underlying these functions is still a growing research field. In bone, the tissue in which BMPs were first discovered, crosstalk of TGFβ/BMP signaling with mechanobiology is well understood. Likewise, the endothelium represents a tissue that is constantly exposed to multiple mechanical triggers, such as wall shear stress, elicited by blood flow or strain, and tension from the surrounding cells and to the extracellular matrix. To integrate mechanical stimuli, the cytoskeleton plays a pivotal role in the transduction of these forces in endothelial cells. Importantly, mechanical forces integrate on several levels of the TGFβ/BMP pathway, such as receptors and SMADs, but also global cell-architecture and nuclear chromatin re-organization. Here, we summarize the current literature on crosstalk mechanisms between biochemical cues elicited by TGFβ/BMP growth factors and mechanical cues, as shear stress or matrix stiffness that collectively orchestrate endothelial function. We focus on the different subcellular compartments in which the forces are sensed and integrated into the TGFβ/BMP growth factor signaling. Since their discovery as cytokines extractable from bone matrix, data on pleiotropic activities by bone morphogenetic proteins (BMPs) belonging to the ligand superfamily of transforming growth factors beta (TGFβs) are vastly expanding. There are more than 30 known TGFβ/BMP ligands, which bind to the heteromeric receptor complexes, comprising two type I R1) and two type II R2) serine/threonine kinase receptors. Numerous co-receptors fine-tune signaling of these receptors [ and two  serine/tBoth TGFβs and BMPs regulate important functions of the vasculature. This is underlined by several vascular phenotypes of the TGFβ/BMP-related knock-outs, which is subject to excellent reviews ,7,8,9. TIn addition to biochemical signals, mechanical signals are equally required to orchestrate blood vessel formation, patterning, branching, pruning, and to maintain their integrity. Crosstalk of TGFβ/BMP signaling with cellular mechanobiology is a growing research field and contribution thereof to the above-mentioned vascular pathologies is little understood. In this review, we focus on the current efforts to understand how biochemical TGFβ/BMP signals in conjunction with mechanical signals, are received and integrated by the endothelium. A special focus is given to the different subcellular compartments in which we and others propose crosstalk to occur.To put endothelial mechanobiology into context, it helps to briefly introduce the different known actions of TGFβ/BMP signaling in the endothelium. ECs can transit between an activated and homeostasis state, regulated by an intricate balance between the activating and homeostatic TGFβ/BMP ligands, in addition to other potent extracellular factors ,14,15; FTGFβ adopts a bipartite role for EC activation vs. homeostasis, dependent on its concentration and engagement of different receptor complexes. TGFβ signals via R1s ALK4 and ALK5. At early stages in the blood vessel development preceding angiogenesis, TGFβ1 mediates vasculogenesis via ALK5 c. Later,In sharp contrast, BMP9 and BMP10, also synthesized as large pro-domain associated precursors, freely circulate in the blood stream ,55, whilWhile several studies report on the anti-angiogenic properties of BMP9, recent studies in human-induced pluripotent stem-cell derived ECs suggest that BMP9 also induces sprouting angiogenesis , which cReceptor regulated (R) SMADs consist of two domains—the MAD homology (MH) 1 domain at the amino-terminus of SMADs is important for their nuclear import and DNA binding. The C-terminal MH2 domain defines R1 binding, SMAD oligomerization, and interaction with cytosolic adaptors and transcriptional co-factors  and Hairy/Enhancer of Split (HES) genes [EC BMP9-SMAD 1/5 signaling upregulates about 100 genes and downregulates around 40 genes more than 2-fold , collectS) genes . AdditioS) genes , suggestAlthough the co-receptor Endoglin is not required for BMP9/10 activation of ALK1, it enhances its signaling output ,79,80,81BMP-SMAD1/5/9 signaling, in particular its turn-over rate, is regulated by endocytosis f, which In the following chapters, we walk through the distinct EC subcellular compartments proposed to incorporate cellular mechanics into the TGFβ/BMP signaling and vice versa . Given iEC junctions b, II resOne main mechanical force coupling to biochemical signals in ECs is the blood-flow generated FSS, which is sensed by the deflection of the primary cilium, for example b, III. EFinally, we conclude our review by placing the EC nucleus into focus, together with the cell’s cytoskeleton as a mechanosensor b, IV. ThThere is a wealth of evidence that actin driven filopodia allow chemotactic and mechanical responses in ECs. The latter include durotaxis, the sensing of stiffness gradients at the tip of the cell, as well as tip-cell pulling forces a that suECM stiffness is beneficial for efficient sprouting angiogenesis in development and disease ,127,128,Mechanical forces involved in sprouting angiogenesis include pulling by the tip-cell and possibly some pushing by the stalk-cells. It was observed through three-dimensional (3D) traction force microscopy that matrix deformations induced by tip-cells were dependent on actin-mediated force generation and correlated with sprout morphological dynamics. Furthermore, sprout tips were found to exert radial pulling forces on the ECM, possibly involving FAs . InteresE) ≤ 5 kPa preferentially deforms under the stresses applied by cells, leaving the latent complex intact and the TGFβ extracellularly sequestered. Whereas a stiff matrix with E >> 10 kPa resists deformation, resulting in distortion of the latent complex and the release of active TGFβ. The crucial role for ECM elasticity and integrin-mediated traction forces is underlined by the finding that absence of integrin-mediated TGFβ1 activation in vivo recapitulates the phenotype of TGFβ1-null mice [Additionally to the regulation of receptor oligomerization, FAs provide a second layer of regulation, through which the mechanics are integrated into endothelial TGFβ/BMP signaling. Integrin-dependent traction forces emerging from FAs directly participate in liberating TGFβ as a biologically active growth factor, from its latent form  c. In detull mice . Mechaniull mice ,159,160.ull mice . Interesull mice , creatinull mice . Mature ull mice ,165,166,ull mice ,168,169.ull mice . TGFβ inull mice  and macrull mice ,172. Morull mice . This suull mice . Fibroneull mice . Latent ull mice ,177. Morull mice . These fSnail, Slug, and Twist genes [Mature ECs are mechanically coupled both to the ECM and via cell junctions to neighboring cells . Howeverst genes ,180,181.It was recently found that force sustained at the cell–cell contact between epithelial cells is approximately 100 nN, directed perpendicular to the cell–cell interface and concentrated at the contact edges . IntriguWe recently showed that angiomotin (AMOT), a TJ-associated membrane protein, drives BMP-SMAD signaling at the apical membrane in polarized cells. In HUVECs, AMOT interacts with BMPR2 and knockdown of AMOT reduces SMAD signaling, only from the apical side of polarized cells, while basolateral BMP-SMAD signaling remained unaffected  Figure a, upper.2) in human vessels [EC phenotype and homeostasis are maintained by a unidirectional, laminar blood flow, ranging approximately from 1 to 5 Pa  p50–p65 heterodimers. Another factor found to bind SSRE is myocyte enhancer factor 2 (MEF2), downstream of MAPK or PI3K signals, which regulates the well-studied shear–stress-induced expression of Krüppel-like factors (KLFs) [The term shear stress response element (SSRE) was first proposed after the identification of the sequence GAGACC, within the shear stress responsive gene promoters , and wass (KLFs) . One mecs (KLFs)  and posss (KLFs) . Global s (KLFs)  and a ´ss (KLFs) . When ECs (KLFs) . Interess (KLFs) .2) leads to decreased lamin A/C expression in ECs [Nuclear import of several transcription factors is altered upon the application of FSS, including YAP . One modn in ECs . Howevern in ECs . Regardin in ECs ,262. In n in ECs ,264. Furn in ECs ,268,269.n in ECs ,270,271.n in ECs . Differen in ECs .Cytoskeletal forces acting on the LINC complex influence heterochromatin localization and core histone protein mobility, and thereby, directly alter gene transcription and induce epigenetic changes d. Transccis or trans to recruit additional enhancers to the SMAD-binding elements. Thus, they increase SMADs transcriptional activity and direct the SMAD complex to particular target genes (CYR61) [CTGF) [A third way through which mechanics integrate into nuclear TGFβ/BMP signaling is through the SMAD dependency on transcriptional co-factors that act in et genes . Two mec (CYR61) ,286 and ) [CTGF) . For BMP) [CTGF) . However) [CTGF) ,288.Slug expression in synergy with TGFβ [A delicate mechanism through which MRTF-A transcription factor integrates into the mechano-dependency of TGFβ/BMP SMAD signaling, is the ability of MRTF-A to directly sense the status of actin remodeling, by binding to globular actin (G-actin) c. MRTF-Aith TGFβ . MRTFs aith TGFβ  and MRTFith TGFβ  and BMP-ith TGFβ , to co-aith TGFβ . Importaith TGFβ . In TGFβith TGFβ . Mechaniith TGFβ , it coulith TGFβ ,297 (FigAs summarized here, crosstalk of endothelial mechanobiology and TGFβ/BMP signaling has a pivotal role for endothelial physiology and pathology. The various mechanisms through which mechanobiology integrate into this delicate signaling network create another layer of regulation that participates in fine-tuning and diversifying TGFβ/BMP signaling. We and others aim to understand the precise molecular mechanisms behind this signaling crosstalk. Such basic molecular investigations remain challenging in an in vivo setting. While previous data gained under static EC culture are a first approximation, technical developments for in vitro tissue culture now allow us to study ECs signaling under biochemical and mechanical conditions, which make these data more relevant to the human system.We have cited examples of how TGFβ/BMP signaling integrates into mechano-transduction in a compartment-specific manner. With crosstalks at discrete membrane domains, the cytoskeleton, the primary cilium, and the nucleus listed, show some of the most crucial compartments integrating EC mechanics into TGFβ/BMP signaling and vice versa. Since investigating endothelial TGFβ/BMP mechano-crosstalk is a relatively young research field, we cited a number of research articles on non-endothelial cells, where we considered as a wealth of evidence that in ECs it might be mechanistically similarly relevant. However, it remains to be proven whether all of the mentioned crosstalk mechanisms were conserved between other cell types and ECs. Further research will help answer these questions and to better understand crosstalk mechanisms of TGFβ/BMP signaling with mechanobiology that underlie human vascular pathologies.


However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7‐like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti‐atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1β at the site of injection. In male mice, CFA‐induced TSLP occurred in immigrated monocytes—and not epithelial cells—and was dependent on NLRP3 inflammasome activation and IL1β‐signalling. In females, CFA‐induced TSLP was independent of IL1β and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR−/− mice, with increased INFγ/IL4 ratio compared with wild‐type controls. To test whether TSLP contributes to the anti‐atherogenic effects of Freund's adjuvant, we treated ApoE−/− and ApoE−/−/TSLPR−/− mice with either CFA/IFA or PBS. ApoE−/− mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE−/−/TSLPR−/− mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune‐modulating effects via TSLP induction. TSLP‐TSLPR signalling is critical for CFA/IFA‐mediated attenuation of atherosclerosis.Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E‐deficient (ApoE Apolipoprotein E‐deficient (ApoE−/−) and RAG1‐deficient (RAG1−/−) mice are originally from the Jax labs. IL1β‐deficient (IL1β−/−) mice were originally kindly provided by Prof. Suda. NLRP3‐deficient (NLRP3−/−) mice were bought from Dr Bernhard Ryffel. ApoE−/−/TSLPR−/− mice were generated by crossing both lines. CCR2‐deficient (CCR2−/−) mice were kindly provided by Dr Christophe Combadiere.C57BL/6 wild‐type mice were purchased from Janvier Labs. TSLPR‐deficient  and were approved by the Institut National de la Santé et de la Recherche Médicale.2.2For immunization experiments Table , the micCirculating monocytes were depleted using liposomes containing clodronate (dichloromethylene diphosphonate) (ClodronateLiposomes.org). Animals received 150 μL intravenous injections of clodronate‐ or PBS‐liposomes. Depletion was verified by flow cytometry.2.33) were placed in 1 mL TRIzol Reagent (Thermo Fisher Scientific) and disrupted with a Polytron homogenizer (Thomas Scientific). Debris was removed through centrifugation for 10 minutes at 7500 g. The supernatant was transferred and then further processed according to the manufacturer's protocol. The RNA pellet was finally dissolved in 100 µL RNAse/DNAse free water and quantified with a NanoDrop. cDNA synthesis was performed with QuantiTect Reverse Transcription Kit (Qiagen).For isolation of RNA from mouse skin, explanted parts  or β‐actin  were used to normalize gene expression. The following primer sequences were used:Quantitative real‐time PCR (polymerase chain reaction) was performed on a StepOnePlus Real‐Time PCR System (Applied Biosystems) using SYBR green PCR master mix (Applied Biosystems). Cycle thresholds for IL1β: F 5′ – GAA GAG CCC ATC CTC TGT GA – 3′, R 5′ – GGG TGT GCC GTC TTT CAT TA – 3′; TSLP: F 5′ – TGA CTG GAG ATT TGA AAG GGG CTA AGT – 3′; R 5′ – CTT GTT CTC CGG GCA AAT GTT TTG T – 3′; IL‐6: F 5′ – AAA GAC AAA GCC AGA GTC CTT CAG AGA GAT – 3′, R 5′ – GGT CTT GGT CCT TAG CCA CTC CTT CTG T – 3′; TNFα: F 5′ – GAT GGG GGG CTT CCA GAA CT – 3′, R 5′ – CGT GGG CTA CAG GCT TGT CAC – 3′; INFβ: F 5′ – CCA CCA CAG CCC TCT CCA TCA ACT AT – 3′, R 5′ – GGA TCT TGA AGT CCG CCC TGT AGG T – 3′; INFγ: F 5′ – AGG AAC TGG CAA AAG GAT GGT GA – 3′; R 5′ – CGC TGG ACC TGT GGG TTG T – 3′.2.5Peripheral blood samples were used for the detection of monocyte subsets upon clodronate treatment. For the analysis of TSLP expression in the skin, excised tissue samples were placed into a cocktail of collagenase I, collagenase XI, DNase I and hyaluronidase (Sigma‐Aldrich); and shaken at 37°C for 1 hour. Cells were then triturated and centrifuged .Cells were labelled with anti – CD11b – FITC or eFluor 450 (eBioscience), anti–Gr1‐PerCP‐Cy5.5 (eBioscience), anti‐CD115‐PE (eBioscience), anti–CD11c‐PE‐Cy7 (eBioscience) or anti–CD45‐FITC (eBioscience) and then analysed by flow cytometry on a Fortessa cytometer (Becton Dickinson). For intracellular cytokine staining, surface staining was performed before permeabilization using an intracellular staining kit (eBioscience). TSLP was labelled with anti‐TSLP‐Alexa Fluor 488 (Bioss Antibodies).2.6Skin samples were directly frozen in OCT at −20° for 1 hour and then transferred to −80°C. Transversal 10 µm sections were thawed and fixed with acetone at 4°C for 10 minutes before further staining according to standard protocols. Briefly, the samples were blocked with 10% FCS for 30 minutes and then incubated with the primary antibody  for 120 minutes at room temperature. After washing with PBS, they were incubated with a secondary antibody for 60 minutes. Nuclei were counterstained with DAPI. Hearts were stored in 4% paraformaldehyde at 4°C. Before inclusion in a cutting medium and further storage at −80°C, hearts were treated with 10% sucrose in phosphate buffered saline for 24 hours. Successive 10 µm transversal sections of aortic sinus were obtained. Lipids were detected using Oil Red O staining, monocytes/macrophages with anti‐MOMA2 (Acris).2.7β‐mercaptoethanol and antibiotics Penicillin and Streptomycin (referred to as “complete” RPMI). ELISpot assays for IFNγ (BD) and IL4 (BD) were performed according to manufacturer's instructions. Cells were incubated for 24 hours (IFNγ) or 48 hours  in complete RPMI in the presence of 100 µg/mL chicken ovalbumin (Sigma) as indicated. For immunoglobulin measurement, a standard ELISA was performed with chicken Ovalbumin coating (1 mg/mL PBS) and goat anti‐mouse IgG1 (SouthernBiotech) or goat anti‐mouse IgG2c (SoutherBiotech).A total of 500 000 spleen cells per well were used, and all samples were used in triplicates. Cells were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, 0.02 mmol/L 2.8t test. Differences were considered statistically significant when the probability value was ≤.05.Data are expressed as mean ± SEM. Data were compared, and intergroup differences were analysed with one‐way ANOVA and post hoc Tukey's test. Other data were analysed by a two‐tailed Student's 33.1−/− mice and found that TSLP was also strongly induced in their skin 12 hours upon s.c. injection of CFA , IL1β was mildly, but statistically significantly increased upon CFA, whereas TSLP was not Figure . In orden Figure . Interesd Figure A‐C. TSLPd Figure .−/− mice, upstream of IL1β production  displayed a comparatively lower IL4 production in TSLPR−/− mice exposition of total splenocytes to OVA, and even though IL4 was still produced to some extent in the absence of TSLPR in males, IFNγ production was dramatically boosted leading to a significant increase of IFNγ/IL4 ratio, and a likewise increase of IgG2c/IgG1 ratio. Thus, our data identify a critical role for TSLP in the modulation of the immune response to Freund's adjuvant. The role of TSLP in promoting a type 2 immune response is consistent with previous observations. TSLP production by DCs fosters a Th2 polarization when interacting with T cells,When we tested the relevance of TSLP/TSLPR signalling in immunization with CFA through antigen exposure, we found that TSLP was crucial to balance IFN−/− mice. Recent studies have addressed the role of TSLP in murine atherosclerosis with partly conflicting results. Wu et al hypothesized a pro‐atherosclerotic role for TSLP, because ApoE−/−TSLPR−/− mice on a high fat diet had less atherosclerosis than ApoE−/− controls.−/− mice fed a high fat and cholesterol rich diet as used by Wu et al, display very severe hypercholesterolaemia and exaggerated innate immune responses,Finally, our study establishes a critical role for TSLPR signalling in CFA/IFA‐mediated inhibition of atherogenesis in ApoEβ‐dependent expression of TSLP. TSLP/TSLPR signalling also mediates the atheroprotective effect of Freund's adjuvant in ApoE−/− mice.In summary, we have shown that TSLP is critically involved in the induction of type 2 immune responses to Freund's adjuvant. In males, this occurs through NLRP3 inflammasome and IL1The authors have no conflicts of interest.MS designed and performed experiments, analysed data, wrote article; LL, SN, LW, BV, PP, BE, MV, AG, CG, ES, TR and SP performed experiments; GN and TR contributed materials and analysis tools; AT conceived and designed experiments, analysed data; Z conceived and designed experiments, contributed materials and analysis tools, analysed data and wrote article; all authors read and approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Fig S5Click here for additional data file.Fig S6Click here for additional data file.Table S1Click here for additional data file.Supplementary MaterialClick here for additional data file.