BMP Pathway Research Articles

The sonic hedgehog signaling pathway and the development of pharyngeal arch Derivatives in Haplochromis piceatus, a Lake Victoria cichlid

ObjectivesPharyngeal arches develop in the head and neck regions, and give rise to teeth, oral jaws, the hyoid bone, operculum, gills, and pharyngeal jaws in teleosts. In this study, the expression patterns of genes in the sonic hedgehog (shh), wnt, ectodysplasin A (eda), and bone morphogenetic protein (bmp) pathways were investigated in the pharyngeal arches of Haplochromis piceatus, one of the Lake Victoria cichlids. Furthermore, the role of the shh pathway in pharyngeal arch development in H. piceatus larvae was investigated. MethodsThe expression patterns of lymphocyte enhancer binding factor 1 (lef1), ectodysplasin A receptor (edar), shh, patched 1 (ptch1), bmp4, sp5 transcription factor (sp5), sclerostin domain containing 1a (sostdc1a), and dickkopf 1 (dkk1) were investigated in H. piceatus larvae by in situ hybridization. The role of the shh pathway was investigated through morphological phenotypic characterization after its inhibition. ResultsWe found that lef1, edar, shh, ptch1, bmp4, dkk1, sostdc1a, and sp5 were expressed not only in the teeth, but also in the operculum and gill filaments of H piceatus larvae. After blocking the shh pathway using cyclopamine, we observed ectopic shh expression and the disappearance of ptch1 expression. After six weeks of cyclopamine treatment, an absence of teeth in the oral upper jaws and a poor outgrowth of premaxilla, operculum, and gill filaments in juvenile H. piceatus were observed. ConclusionsThese results suggest that the shh pathway is important for the development of pharyngeal arch derivatives such as teeth, premaxilla, operculum, and gill filaments in H. piceatus.

Inhibition of Notch Signaling During Mouse Incisor Renewal Leads to Enamel Defects.

The continuously growing rodent incisor is an emerging model for the study of renewal of mineralized tissues by adult stem cells. Although the Bmp, Fgf, Shh, and Wnt pathways have been studied in this organ previously, relatively little is known about the role of Notch signaling during incisor renewal. Notch signaling components are expressed in enamel-forming ameloblasts and the underlying stratum intermedium (SI), which suggested distinct roles in incisor renewal and enamel mineralization. Here, we injected adult mice with inhibitory antibodies against several components of the Notch pathway. This blockade led to defects in the interaction between ameloblasts and the SI cells, which ultimately affected enamel formation. Furthermore, Notch signaling inhibition led to the downregulation of desmosome-specific proteins such as PERP and desmoplakin, consistent with the importance of desmosomes in the integrity of ameloblast-SI attachment and enamel formation. Together, our data demonstrate that Notch signaling is critical for proper enamel formation during incisor renewal, in part by regulating desmosome-specific components, and that the mouse incisor provides a model system to dissect Jag-Notch signaling mechanisms in the context of mineralized tissue renewal.

OC-008 A 3D primary tumour drug screen identifies hedgehog signalling as a key regulator of intestinal cancer stem cell quiescence and tumorigenesis

<h3>Introduction</h3> Tumour dormancy and cellular quiescence have recently been strongly implicated in disease recurrence after adjuvant therapy in colorectal cancer (CRC). CRC is increasingly recognised as a heterogeneous disease with significant implications for medical and surgical management. Little is known about the control of cellular quiescence and the molecular features of dormant cells in CRC. Here we present the molecular and functional features of dormant intestinal tumour cells and describe the paracrine signalling pathways that govern their behaviour. <h3>Method</h3> Transgenic Apc^1322t/H2BYFP mice were generated to allow the characterisation, both functionally and molecularly of dormant tumour cells. A drug screen was performed on <i><i>ex vivo</i></i>3D matrigel-cultured tumour spheroids. Proliferation kinetics, growth characteristics and molecular alterations were correlated with known signalling pathways implicated in intestinal tumorigenesis. Selected compounds were then advanced to validation using six human colorectal cancer cell lines, that represent the three main subtypes of CRC, in 2D and 3D culture experiments. <h3>Results</h3> Dormant tumour cells represent a small population of tumour cells that are highly clonogenic, chemoresistant and share many features similar to reserve intestinal stem cells. The Hedgehog and BMP pathways play a dominant role in controlling the growth of tumour spheroids in culture. Antagonism of the Hedgehog pathway causes spheroid growth collapse, the activation of dormant tumour cells and yet no cytotoxicity. The effects of Hedgehog antagonism appears to be CRC sub-type specific; with the most marked effects in the commonest CCSN1 (CIN) subtype of CRC. <h3>Conclusion</h3> Here we provide the first molecular and functional characterisation of dormant tumour cells, showing them to represent the hypothesised quiescent cancer stem cell. Interestingly, our data confirms that of others, in demonstrating Wnt inactivation fails to effect the growth of Apc-mutant tumour cells. However we show that the Hedgehog pathway appears to control not only spheroid growth at a global level but also and uniquely the activation of dormant tumour cells. Manipulation of the Hedgehog pathway in combination with standard-of-care chemotherapy may provide significant benefits for patients with classical Apc-mutated and Wnt driven CRC. <h3>Disclosure of interest</h3> None Declared. <h3>References</h3> Buczacki SJ, Zecchini HI, Nicholson AM, <i>et al</i>. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 2013;495(7439):65–9 Buczacki SJA, Davies RJ. The confounding effects of tumour heterogeneity and cellular plasticity on personalized surgical management of colorectal cancer. Colorectal Dis. 2014;16(5):329–31

Smad1/5 and Smad4 expression are important for osteoclast differentiation.

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP's effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity.

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.

BMP-2 promoted glioma cell proliferation via regulation of BMP/Smad and BMP/MAPK pathway

Objective To investigate the BMP-2 expression in glioma cell lines and its promotive role and mechanism of proliferation in glioma cells. Methods Subculture of five glioma cell lines including U8vIII, U87, T98G, LN229 and U373, Semi-quantitative PCR was performed to analyze BMP-2, BMP-2 receptors (BMPR1A, BMPR1B and Alk-2) and Smad1 expression in these glioma cell lines. MTS assay was used to investigate the glioma cell proliferation with BMP-2 (0, 25, 50, 100, 250 and 500 ng/ml) treatment. Western blot was performed to detect activation of canonical BMP pathway (BMP/Smad pathway) and non-canonical BMP pathway (BMP/MAPK pathway) under BMP-2 treatment with different doses and timepoints. At last, the expression of BMP pathway downstream genes cyclin B, cyclin D1, p27 and p21 were explored at 0, 4, 8, 16 or 24 h posttreatment of BMP-2 (100 ng/ml). Results The expression of BMP-2, its receptors and Smad1 were significantly different among five glioma cell lines (P<0.01). The MTS assay showed that BMP-2 promoted U87 cell proliferation. It reached the maximum level when the BMP-2 concentration reached 100-250 ng/ml. The Western blot results revealed that BMP-2 activated phosphorylated Smad1/5/8, p38, JNK and ERK in dose and time dependent manners (P<0.01). The expression levels of BMP pathway downstream genes cyclin B and cyclin D1 were higher compared to those of control (P<0.01). However, the levels of p27 and p21 were lower (P<0.01). Conclusions BMP-2 could promote glioma cell proliferation via regulation of BMP/Smad and BMP/MAPK pathway. Key words: Glioma; Bone morphogenetic protein 2; Mitogen-activated protein kinases; Smad proteins

On the development of the patella.

The current view of skeletal patterning fails to explain the formation of sesamoid bones. These small bones, which facilitate musculoskeletal function, are exceptionally embedded within tendons. Although their structural design has long puzzled researchers, only a limited model for sesamoid bone development has emerged. To date, sesamoids are thought to develop inside tendons in response to mechanical signals from the attaching muscles. However, this widely accepted model has lacked substantiation. Here, we show that, contrary to the current view, in the mouse embryo the patella initially develops as a bony process at the anteriodistal surface of the femur. Later, the patella is separated from the femur by a joint formation process that is regulated by mechanical load. Concurrently, the patella becomes superficially embedded within the quadriceps tendon. At the cellular level, we show that, similar to bone eminences, the patella is formed secondarily by a distinct pool of Sox9- and Scx-positive progenitor cells. Finally, we show that TGFβ signaling is necessary for the specification of patella progenitors, whereas the BMP4 pathway is required for their differentiation. These findings establish an alternative model for patella development and provide the mechanical and molecular mechanisms that underlie this process. More broadly, our finding that activation of a joint formation program can be used to switch between the formation of bony processes and of new auxiliary bones provides a new perspective on plasticity during skeletal patterning and evolution.

Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

Synthesis of a novel class of substituted benzothiophene or benzofuran derivatives as BMP-2 up-regulators and evaluation of the BMP-2-up-regulating effects in vitro and the effects on glucocorticoid-induced osteoporosis in rats

The BMP pathway is a promising new target for the design of therapeutic agents for the treatment of low bone mass. To enrich our understanding of SAR and based on our previously concluded structure–effect relationship, 23 derivatives were prepared in this work. The synthesis, up-regulating activities on BMP-2 expression, and bone loss prevention efficacies of these compounds in rats with glucocorticoid-induced osteoporosis are presented. The bone histology of the tested rats assessed through light microscopy showed that compounds 1, 21, 35, and 38 significantly increased the trabecula compared with the model group, and the trabecula of the groups treated with 8a was similar to that obtained with raloxifene and alfacalcidol. The compounds exhibited potential for development as anabolic agents.

Improving the osteogenic efficacy of BMP2 with mechano growth factor by regulating the signaling events in BMP pathway.

Local application of bone morphogenetic protein 2 (BMP2) is known to promote large bone defect healing and BMP2-initiated bone regeneration could be enhanced by an additional mechanical stimulation. The C-terminal 24-a.a. peptide of mechano growth factor (MGF24E), a mechanical-sensitive molecule, has been demonstrated to promote bone healing. Here, we propose a hypothesis that MGF24E could also improve the osteogenic efficacy of BMP2 by regulating the signaling events in the BMP pathway. To confirm the hypothesis, the potentials of MGF24E, BMP2 and BMP2/MGF24E combination treatments on the phosphorylation of Smad 1/5/8, the downstream osteogenesis-related gene expression and osteoblasts mineralization, are investigated with or without the blocking of Smad 5 siRNA. Furthermore, 15-mm rabbit radial bone defects were healed with the cytokine treatments and then evaluated by radiographic examination, histological assessment and immunohistochemical analysis. MGF24E could enhance the BMP2-induced Smad signaling pathway by upregulating the p-Smad protein expression and the downstream osteogenic gene expression. An amount of 5 nM BMP2 in a sub-25 nM concentration of MGF24E medium achieved a higher expression for ALP mRNA and a greater calcium mineral content compared with BMP2 alone. Nevertheless, the inhibition of the MGF24E-regulated BMP pathway could block osteogenesis induced by the dual treatment. In vivo, MGF24E treatment upregulated the endogenous BMP2 expression and the addition of MGF24E into the BMP2 treatment remarkably enhanced the bone mineral density (BMD), the radiographic scores and the histological restoration of the regenerated tissue against BMP2 treatment, suggesting a new strategy for BMP2 in bone defect healing.

The DOT1L protein and gene network in chondrocytes identifies H3K79 histone methylation as a key regulator of WNT and other growth factor cascades

Purpose: DOT1L is the only known H3K79 histone methyltransferase and has been linked to epigenetic regulation of the Wingless-like (Wnt) signaling pathway. Genome-wide association and functional studies identified the DOT1L gene to be associated with cartilage thickness and hip osteoarthritis (OA) and showed an interaction of DOT1L with canonical Wnt signaling. Here, we further investigated the biology of DOT1L in cartilage health and disease. Specifically, our objective was to define the transcriptional complexes and transcriptome associated with DOT1L in articular cartilage, and to link these DOT1L targets and interactors with the molecular characteristics of the articular chondrocyte during the loss of phenotype in a dedifferentiation setup. Finally, we aimed for a translational validation of our data in cartilage samples from OA patients. Methods: Human articular chondrocytes (hACs) stimulated or not with LiCl were used to carefully map the presence or absence of DOT1L protein complexes suggested earlier in leukemia cells, using a series of specific immunoprecipitation experiments. To elucidate the transcriptional network of Dot1l, we performed a microarray of hACs from 5 non-OA fracture patients treated with a specific DOT1L inhibitor (EPZ5676) or vehicle control. The differentially expressed genes were explored with the limma package. String-DB and Panther were used for gene network analyses. Based on the microarray analysis, selected genes were confirmed by quantitative PCR. To establish the role of DOT1L in the maintenance of hACs phenotype, hACs were cultured in monolayer in the presence or absence of EPZ5676, and RNA and protein were isolated at serial passages (P0 to P5). We analyzed by quantitative PCR the gene expression of known genes important for chondrocyte biology and/or genes that appeared in the microarray of DOT1L inhibition. Activation of the Wnt canonical and non-canonical signaling cascades was analyzed by Western blot. For translational validation we analyzed the differential gene expression profile of damaged as compared to intact cartilage areas within the same joint of patients with hip OA. Results: The presence of different DOT1L elongation complexes was confirmed in hACs. The CoIP experiments revealed that DOT1L interacts directly with ENL, AF10 and active beta-catenin (more strongly when cells were treated with LiCl to stabilize beta-catenin) and with AF4 and AF5 (only with cells were treated with LiCl). The formation of these complexes was disrupted when DOT1L was inhibited. In the microarray, 1937 genes significantly changed (p<0.05) in DOT1L inhibited samples compared to vehicle treated samples (1048 genes were down-regulated, 889 up-regulated). In EPZ5676 treated samples, chondrocyte differentiation-associated genes, such as ACAN, RGS5, GDF10 and LOXL2 were down-regulated; OA-related genes, such as MMP1, CCL7, CCL8 and GPNMB were up-regulated; and WNT target genes, ligands and antagonists such as LEF1, WNT5A and DKK1 were significantly up-regulated. In the de-differentiation experiment, in vitro expanded hACs showed progressive changes in gene expression. Genes involved in relevant pathways for cartilage biology such as the WNT, NOTCH and BMP pathways exhibited significant changes that were highly accentuated by DOT1L inhibition. We successfully validated these targets of interest in samples from patients with OA condition. Conclusions: Our transcriptomic, protein and gene interaction approach provides novel insights into the DOT1L molecular network and its putative role in osteoarthritis and cartilage. These data further support an important role for DOT1L in joint homeostasis as a key regulator of WNT signaling and other growth factor cascades in the joint.

Rat aortic smooth muscle cells cultured on hydroxyapatite differentiate into osteoblast-like cells via BMP-2-SMAD-5 pathway.

Vascular calcification is an important pathological condition associated with increased risk of cardiovascular mortality. Hydroxyapatite (HA) found in such deposits is the same polymorph of calcium (Ca) found in bone, indicating calcification may involve mechanisms akin to bone formation. Vascular smooth muscle cells (Vsmcs) have been shown to undergo phenotypic change to osteoblast-like cells. However, the mechanisms underlying this phenotypic change are unclear, and whether the stimulus to become osteogenic is a result of loss of mineralization inhibitors or early mineral deposits is not known. Our aim in this study is to identify mechanisms and signal transduction pathways that cause differentiation of Vsmcs into osteoblast-like cells in the presence of HA. We first characterized vascular origin of Vsmcs by studying the expression of smooth muscle cell markers: myosin heavy chain and smooth muscle actin along with SM22α at both mRNA and protein levels. Vsmcs grown on HA exhibited progressive change in cellular morphology at 3-, 7-, and 14-day time points. Culturing of Vsmcs on HA disc resulted in decrease in media Ca levels and increased expression of Ca-sensing receptor (CaSR) on Vsmcs resulting inupregulation of intracellular CaSR signaling leading to increased BMP-2 secretion. BMP-2 pathway mediated differentiation of Vsmcs to osteoblast-like cells shown by expression of osteogenic markers like runt-related transcription factor 2, osteocalcin, and alkaline phosphatase at mRNA and protein levels. Blocking CaSR by NPS-2143 reduced BMP-2 secretion and blocking the BMP-2 pathway by LDN-193189, a BMP inhibitor, modulated expression of osteogenic markers confirming their role in osteogenesis of Vsmcs.

Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis.

The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0–1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50–100 ng/ml) during Day 0–1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3–10 ng/ml) of Bmp4 during Day 0–1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.

Regulatory expression of components in the BMP pathway in white adipose tissues of cattle

The BMP pathway positively regulates murine brown adipogenesis. We herein examined the mRNA levels of BMPs and activin βB as well as receptors for the BMP pathway in the adipose tissues of cattle fed diets with a differential ratio of concentrate to roughage or a vitamin A-deficient diet. The expression of activin βB was significantly increased in the subcutaneous fat depot of animals fed the concentrate diet, while the vitamin A-deficient diet significantly increased the expression of BMP4 in the mesenteric fat depot. The expression of receptors for the BMP pathway, ALK2, ALK3, ActRIIA, and BMPR2, showed a similar pattern to that of BMP4 and activin βB in response to the dietary treatments. The results of the present study demonstrated that diet modulated the expression of components of the BMP pathway and may be responsible for the regulatory expression of brown/beige adipocyte-related genes in the adipose tissues of cattle.

Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron.

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

MTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration

Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration.

Transcriptional Pathways Associated with Skeletal Muscle Changes after Spinal Cord Injury and Treadmill Locomotor Training.

The genetic and molecular events associated with changes in muscle mass and function after SCI and after the implementation of candidate therapeutic approaches are still not completely known. The overall objective of this study was to identify key molecular pathways activated with muscle remodeling after SCI and locomotor training. We implemented treadmill training in a well-characterized rat model of moderate SCI and performed genome wide expression profiling on soleus muscles at multiple time points: 3, 8, and 14 days after SCI. We found that the activity of the protein ubiquitination and mitochondrial function related pathways was altered with SCI and corrected with treadmill training. The BMP pathway was differentially activated with early treadmill training as shown by Ingenuity Pathway Analysis. The expression of several muscle mass regulators was modulated by treadmill training, including Fst, Jun, Bmpr2, Actr2b, and Smad3. In addition, key players in fatty acids metabolism (Lpl and Fabp3) responded to both SCI induced inactivity and reloading with training. The decrease in Smad3 and Fst early after the initiation of treadmill training was confirmed by RT-PCR. Our data suggest that TGFβ/Smad3 signaling may be mainly involved in the decrease in muscle mass observed with SCI, while the BMP pathway was activated with treadmill training.

BMP signalling controls the construction of vertebrate mucociliary epithelia.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.

Neurotrophine-3 may contribute to neuronal differentiation of mesenchymal stem cells through the activation of the bone morphogenetic protein pathway.

We investigated whether neurotrophin-3 (NT-3) can promote differentiation of mouse bone mesenchymal stem cells (MSCs) into neurons via the bone morphogenetic protein pathway. MSCs were prepared from rat bone marrow and either transfected with pIRES2-EGFP or pIRES2-EGFP-NT-3 or treated with bone morphogenetic protein 4. The pIRES2-EGFP-NT-3-transfected MSCs further underwent noggin treatment or siRNA-mediated knockout of the TrkC gene or were left untreated. Immunofluorescence staining, real-time PCR and Western blot analyses were performed to evaluate the transcription and expression of neural-specific genes and BMP-Smad signaling. MSCs were efficiently transduced by the NT-3 gene via pIRES2-EGFP vectors. pIRES2- EGFP-NT-3 could initiate the transcription and expression of neural-specific genes, including nestin, NSE and MAP-2, and stimulate BMP-Smad signaling. The transcription and expression of neural-specific genes and BMP-Smad signaling were significantly suppressed by siRNA-mediated knockdown of the TrkC gene of MSCs. These findings suggest that the BMP signaling pathway may be a key regulatory point in NT-3-transfected neuronal differentiation of MSCs. The BMP and neurotrophin pathways contribute to a tightly regulated signaling network that directs the precise connections between neuronal differentiation of MSCs and their targets.

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues.