The Hedgehog (Hh) pathway is essential for normal embryonic development and tissue repair. The role of Hh signaling in hematopoiesis has been studied primarily by modulating the activity of Patched and Smoothened, but results have been conflicting. Some studies demonstrate a requirement for pathway activity in hematopoiesis, whereas others report that it is dispensable. Hh activity converges on the Gli transcription factors, but the specific role of these downstream effectors in hematopoiesis has not been reported. We have analyzed hematopoietic stem cell (HSC) and progenitor function in mice with a homozygous deletion of Gli1 (Gli1 null ). Gli1 null mice have more longterm HSCs that are more quiescent and show increased engraftment after transplantation. In contrast, myeloid development is adversely affected with decreased in vitro colony formation, decreased in vivo response to granulocyte colonystimulating factor (G-CSF), and impaired leukocyte recovery after chemotherapy. Levels of the proto-oncogene Cyclin D1 are reduced in Gli1 null mice and may explain the loss of proliferation seen in HSCs and progenitor cells. These data demonstrate that Gli1 regulates normal and stress hematopoiesis. Moreover, they suggest that Gli1 and Smoothened may not be functionally redundant, and direct GLI1 inhibitors may be needed to effectively block HH/GLI1 activity in human disease. (Blood. 2010;115:2391-2396) IntroductionThe proliferation of hematopoietic stem cells (HSCs) and progenitors is tightly regulated during normal homeostasis. HSCs are normally quiescent in the adult mouse but they can be induced to proliferate in response to stress or cytokine stimulation. In contrast, progenitors are highly proliferative to maintain a constant supply of infection-fighting white blood cells. Precisely how HSC and progenitor proliferation are regulated is not completely understood, but recent data have implicated a role for developmental signaling pathways such as Wnt and Notch in the regulation of stem cell proliferation, self-renewal, and differentiation. [1][2][3][4] The Hedgehog (Hh) signaling pathway in mammals consists of 3 closely related ligands, Sonic Hh (Shh), Indian Hh (Ihh), and Desert Hh (Dhh), that can each bind to the transmembrane protein Patched (Ptch). Upon ligand binding, Ptch inhibition of the positive effector Smoothened (Smo) is released and signaling is transduced. Three zinc finger transcription factors, Gli1, Gli2, and Gli3, lie downstream of Smo and mediate Hh's effects. Gli1 is a positive effector of signaling, Gli3 is predominantly a transcriptional inhibitor, and Gli2 can function in both roles. 5 The precise role of Hh signaling in normal hematopoiesis, however, is not known and the literature is contradictory. One group has reported that loss of Smo activity leads to a severe defect in HSC function, 6 whereas others have reported a more modest phenotype, 7 or none at all. 8,9 All of these studies have focused primarily on the upstream modulators of pathway activity, Ptch and Smo. To better understand ...

There is a lack of pharmacological interventions available for sarcopenia, a progressive age-associated loss of muscle mass, leading to a decline in mobility and quality of life. We found mTORC1 (mammalian target of rapamycin complex 1), a well-established positive modulator of muscle mass, to be surprisingly hyperactivated in sarcopenic muscle. Furthermore, partial inhibition of the mTORC1 pathway counteracted sarcopenia, as determined by observing an increase in muscle mass and fiber type cross-sectional area in select muscle groups, again surprising because mTORC1 signaling has been shown to be required for skeletal muscle mass gains in some models of hypertrophy. Additionally, several genes related to senescence were downregulated and gene expression indicators of neuromuscular junction denervation were diminished using a low dose of a “rapalog” (a pharmacological agent related to rapamycin). Therefore, partial mTORC1 inhibition may delay the progression of sarcopenia by directly and indirectly modulating multiple age-associated pathways, implicating mTORC1 as a therapeutic target to treat sarcopenia.

Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila. We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.KEYWORDS Pak, cell adhesion, cell differentiation, myogenesis, regeneration, signal transduction C ell differentiation is a complex process whereby precursor cells take on tissuespecific structure and function. Lineage-restricted transcription factors lie at the heart of cell differentiation, but the process is often initiated and fortified by ubiquitous signaling pathways that function in many biological contexts. Skeletal myogenesis serves as a paradigm for cell differentiation. Differentiation of skeletal myoblasts is a coordinated process involving adoption of a cell-type-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers (1, 2). MyoD family proteins (MyoD, Myf5, myogenin, and MRF4) are muscle-specific transcription factors that act in concert with other, more broadly expressed transcription factors to establish the muscle phenotype (1, 3). The activities of these factors are regulated posttranslationally by non-muscle-specific signal transduction pathways. One such pathway is the p38␣/␤ mitogen-activated protein kinase (MAPK; here simply p38) pathway (4). p38 is activated during myogenic differentiation in vitro, and its inhibition results in impaired differentiation (5-7). Furthermore, mice lacking p38␣ exhibit delayed myofiber growth and maturation (8).The signals that initiate p38 activity during myoblast differentiation are poorly understood. One mechanism is via activation of a signaling complex nucleated at sites of cadherin-based cell-cell adhesion (9, 10). The transmembrane IgSF coreceptor Cdo (also called Cdon) is bound in cis to N-cadherin (Ncad) in myoblasts. During myoblast differentiation, or acutely upon Ncad ligation, the Cdo intracellular region associates directly with (i) Bnip-2, a scaffold protein for Cdc42, and (ii) JLP, a scaffo...

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Muscle-specific genetic ablation of p21-activated kinase (PAK)2, but not whole-body PAK1 knockout, impairs glucose tolerance in mice. r Insulin-stimulated glucose uptake partly relies on PAK2 in glycolytic extensor digitorum longus muscle r By contrast to previous reports, PAK1 is dispensable for insulin-stimulated glucose uptake in mouse muscle.

Background Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. Methods We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation. Results dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. Conclusions Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes. Electronic supplementary material The online version of this article (10.1186/s13395-019-0191-4) contains supplementary material, which is available to authorized users.

Non-standard abbreviations24 2DG: 2-Deoxyglucose 25 AUC: Area under the curve 26 BCA: Bicinchoninic acid 27 BW: Body weight 28 dKO: Double knockout 29 EDL: Extensor digitorum longus 30 FM: Fat mass 31 GLUT4: Glucose transporter 4 32 GTT: Glucose tolerance test 33 HFD: High-fat diet 34 HOMA-IR: Homeostatic Model Assessment of Insulin Resistance 35 ITT: Insulin tolerance test 36 i.p.: Intraperitoneal 37 KO: Knockout 38 L6-GLUT4myc: Rat L6 skeletal muscle cells overexpressing myc-tagged GLUT4 39 LBM: Lean body mass 40 mKO: Muscle-specific knockout 41 NOX: NADPH oxidase 42 PAK: p21-activated kinase 43 PI3K: Phosphoinositide 3-kinase 44 RER: Respiratory exchange ratio 45 r.o.: Retro-orbital 46 VO 2 : Oxygen uptake 47 48Glucose transport into skeletal muscle is essential for maintaining whole-body glucose homeostasis 49 and accounts for the majority of glucose disposal in response to insulin. The group I p21-activated 50 kinase (PAK) isoforms PAK1 and PAK2 are in skeletal muscle activated in response to insulin and 51 evidence suggests that PAK1 is necessary for insulin-stimulated GLUT4 translocation. In 52 accordance, insulin-induced PAK1 and PAK2 signalling are impaired in insulin-resistant skeletal 53 muscle. However, the role of PAK1 and PAK2 in insulin-stimulated glucose uptake in mature 54 skeletal muscle has not been determined. The aim of the present investigation was to determine the 55 requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated 56 glucose uptake in skeletal muscle. Therefore, glucose uptake was measured in isolated skeletal 57 muscle incubated with a pharmacological inhibitor (IPA-3) of group I PAKs and in muscle from 58 whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO and double whole-body PAK1 59 and muscle-specific PAK2 knockout mice. Whole-body respiratory exchange ratio, indicative of 60 substrate utilization, was largely unaffected by lack of PAK1 and/or PAK2. Whole-body glucose 61 tolerance was mildly impaired in PAK2 mKO, but not PAK1 KO mice. In contrast to a previous 62 study of GLUT4 translocation in PAK1 KO mice, PAK1 KO muscles displayed normal insulin-63 stimulated glucose uptake in vivo and in isolated muscle. On the contrary, glucose uptake was 64 slightly reduced in response to insulin in glycolytic extensor digitorum longus muscle lacking 65 PAK2. In conclusion, the current study demonstrates that group I PAKs are largely dispensable for 66 the regulation of whole-body glucose homeostasis and skeletal muscle glucose uptake. Thus, the 67 present study challenges that group I PAKs, and especially PAK1, are necessary regulators of 68 insulin-stimulated glucose uptake in skeletal muscle. 69 109Although such studies implicate group I PAKs, and in particular PAK1, in the regulation of glucose 110 homeostasis and GLUT4 translocation in skeletal muscle, the relative role of PAK1 and PAK2 in 111 insulin-stimulated glucose uptake remains to be identified in mature skeletal muscle. Therefore, we 112 performed a systematic series of pharmacologic a...

12There is a lack of pharmacological interventions available for sarcopenia, a progressive age-13 associated loss of muscle mass, leading to a decline in mobility and quality of life. We found 14 mTORC1 (mammalian target of rapamycin complex 1), a well-established critical positive 15 modulator of mass, to be hyperactivated in sarcopenic muscle. Furthermore, inhibition of the 16 mTORC1 pathway counteracted sarcopenia as determined by observing an increase in muscle 17 mass and fiber type cross sectional area, surprising because mTORC1 signaling has been shown 18 to be required for muscle mass gains in some settings. Additionally, several genes related to 19 senescence were downregulated, while gene expression indicators of neuromuscular junction 20 denervation were diminished using a low dose of a rapalog. Therefore mTORC1 inhibition may 21 delay the progression of sarcopenia by directly and indirectly modulating multiple age-associated 22 pathways, implicating mTORC1 as a therapeutic target to treat sarcopenia. 23 29 decrease in walking speed is one of the strongest predictors of mortality in humans, and this 30 finding is associated with sarcopenia (3, 4). In addition to frailty and sarcopenia, aging of course 31 affects every tissue system and greatly increases susceptibility to other serious diseases and co-32 morbidities, such as cancer, heart failure, chronic kidney disease, loss of vision, dementia and 33 Alzheimer's disease (1, 5, 6). 34Experimental data strongly suggest the coordinated regulation of aging by distinct 35 molecular pathways (7); modulation of these pathways can counteract several age-related 36 diseases and co-morbidities, and prolong life (7-10). Of these signaling pathways, genetic or 37 pharmacological inhibition of the mammalian target of rapamycin (mTORC1) is thus far the 38 best-validated intervention to delay age-related pathophysiological changes (11). For instance, 39 the use of an mTORC1 inhibitor, rapamycin, even when administered at later stages in life, has 40 been shown to extend lifespan in mice (12)(13)(14)(15). Pharmalogical agents related to rapamycin are 41 called "rapalogs". Use of a rapalog for aging-like indications has recently been translated to 42 human beings, where it was shown to improve responses to vaccinations in the elderly, 43 coincident with decreasing signs of immune-senescence (16). The low dose rapalog treatment 44 used in the human study was reverse-translated to rats, where it was shown that intervention late 45 in life could prevent signs of age-related kidney pathology (17). However, there has always been 46 4 concern about the potential effects of rapamycin and rapalogs on skeletal muscle. For example, 47 inhibition of the mTORC1 pathway was shown to entirely block responses to compensatory 48 hypertrophy in mice (18). This certainly gave the impression that activation of mTORC1 49 signaling was desireable for the maintenance of muscle mass. Most recently it was shown that 50 rapamcyin treatment inhibited muscle mass increase caused by myostatin lo...