Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Activin Controls Ethanol Potentiation of Inhibitory Synaptic Transmission Through GABAA Receptors and Concomitant Behavioral Sedation.

Activin, a member of the transforming growth factor-ß family, exerts multiple functions in the nervous system. Originally identified as a neurotrophic and -protective agent, increasing evidence implicates activin also in the regulation of glutamatergic and GABAergic neurotransmission in brain regions associated with cognitive and affective functions. To explore how activin impacts on ethanol potentiation of GABA synapses and related behavioral paradigms, we used an established transgenic model of disrupted activin receptor signaling, in which mice express a dominant-negative activin receptor IB mutant (dnActRIB) under the control of the CaMKIIα promoter. Comparison of GABAA receptor currents in hippocampal neurons from dnActRIB mice and wild-type mice showed that all concentrations of ethanol tested (30-150 mM) produced much stronger potentiation of phasic inhibition in the mutant preparation. In dentate granule cells of dnActRIB mice, tonic GABA inhibition was more pronounced than in wild-type neurons, but remained insensitive to low ethanol (30 mM) in both preparations. The heightened ethanol sensitivity of phasic inhibition in mutant hippocampi resulted from both pre- and postsynaptic mechanisms, the latter probably involving PKCɛ. At the behavioral level, ethanol produced significantly stronger sedation in dnActRIB mice than in wild-type mice, but did not affect consumption of ethanol or escalation after withdrawal. We link the abnormal narcotic response of dnActRIB mice to ethanol to the excessive potentiation of inhibitory neurotransmission. Our study suggests that activin counteracts oversedation from ethanol by curtailing its augmenting effect at GABA synapses.

Context-specific interactions between Notch and ALK1 cannot explain ALK1-associated arteriovenous malformations.

AIMS: Notch and activin receptor-like kinase 1 (ALK1) have been implicated in arterial specification, angiogenic tip/stalk cell differentiation, and development of arteriovenous malformations (AVMs), and ALK1 can cooperate with Notch to up-regulate expression of Notch target genes in cultured endothelial cells. These findings suggest that Notch and ALK1 might collaboratively program arterial identity and prevent AVMs. We therefore sought to investigate the interaction between Notch and Alk1 signalling in the developing vertebrate vasculature. METHODS AND RESULTS: We modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Although Alk1 is not necessary for expression of Notch target genes in arterial endothelium, loss of Notch signalling unmasks a role for Alk1 in supporting hey2 and ephrinb2a expression in the dorsal aorta. In contrast, Notch and Alk1 play opposing roles in hey2 expression in cranial arteries and dll4 expression in all arterial endothelium, with Notch inducing and Alk1 repressing these genes. Although alk1 loss increases expression of dll4, AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Dll4/Notch inhibition. CONCLUSION: Control of Notch targets in arterial endothelium is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Alk1 is not required for arterial identity, and perturbations in Notch signalling cannot account for alk1 mutant-associated AVMs. These data suggest that AVMs associated with ALK1 mutation are not caused by defective arterial specification or altered Notch signalling.

The role of Alk-1 and Alk-5 in the mechanosensing of chondrocytes.

We aim to demonstrate the role of Alk receptors in the response of hydrogel expansion. Chondrocytes from rat knees were cultured onto plastic and hydrogel surfaces. Alk-1 and Alk-5 were overexpressed or silenced and the effects on cells during expansion were tested and confirmed using peptide inhibitors for TGFß. Overexpression of Alk-5 and silencing of Alk-1 led to a loss of the chondrocyte phenotype, proving that they are key regulators of chondrocyte mechanosensing. An analysis of the gene expression profile during the expansion of these modified cartilage cells in plastic showed a better maintenance of the chondrocyte phenotype, at least during the first passages. These passages were also assayed in a mouse model of intramuscular chondrogenesis. Our findings indicate that these two receptors are important mediators in the response of chondrocytes to changes in the mechanical environment, making them suitable targets for modulating chondrogenesis. Inhibition of TGFß could also be effective in improving chondrocyte activity in aged or expanded cells that overexpress Alk-1.

Activin receptor-like kinase and the insulin gene.

The biological responses of the transforming growth factor-ß (TGF-ß) superfamily, which includes Activins and Nodal, are induced by activation of a receptor complex and Smads. A type I receptor, which is a component of the complex, is known as an activin receptor-like kinase (ALK); currently seven ALKs (ALK1-ALK7) have been identified in humans. Activins signaling, which is mediated by ALK4 and 7 together with ActRIIA and IIB, plays a critical role in glucose-stimulated insulin secretion, development/neogenesis, and glucose homeostatic control of pancreatic endocrine cells; the insulin gene is regulated by these signaling pathways via ALK7, which is a receptor for Activins AB and B and Nodal. This review discusses signal transduction of ALKs in pancreatic endocrine cells and the role of ALKs in insulin gene regulation.

[Activin and activin receptors in related gynecologic and obstetric diseases].

Activin is a member of the transforming growth factor-beta (TGF-beta) superfamily that result from the assembly of disulphide-linked betaA and betaB subunits. Activin receptors are transmembrane proteins and activin fulfils the biological function through the signal transduction of the receptor system. In recent years, many studies have suggested that activins have wide biological activities. It is the basic medium in regulating histiocytic function and plays a role in maintaining the normal function of cells. Moreover, abnormal expression of activin in the tissues of many gynecologic and obstetric diseases, such as epithelial ovarian tumor, endometrial carcinoma, pre-eclampsia, polycystic ovary syndrome, endometriosis and so on affects the development of these diseases.

FoxL2 Is required for activin induction of the mouse and human follicle-stimulating hormone beta-subunit genes.

Activin is a major physiological regulator of FSH. We identify FoxL2 as a critical component in activin induction of FSHbeta, both for the mouse gene, induction of which is Sma- and Mad-related protein (Smad) dependent, and for the human gene that is Smad independent. FoxL2 has been shown to regulate gonadotrope gene expression (GnRH receptor, alpha-glycoprotein subunit, porcine FSHbeta, and follistatin), but the mechanisms of action are not well understood. We identify novel sites required for activin action in both the mouse and human FSHbeta promoters, some of which bind FoxL2, and show that the FoxL2-binding element encompasses a larger region (12 bp) than the previously identified forkhead-binding consensus (7 bp). Remarkably, although required for activin induction, FoxL2 sites neither contribute to basal FSHbeta promoter activity nor confer activin response to a heterologous promoter; thus, they are neither classical activin-response elements nor is their role solely to recruit Smads to the promoter. FoxL2 overexpression can potentiate activin induction in gonadotropes and can confer activin responsiveness to FSHbeta in heterologous cells where this promoter is normally refractory to activin induction. Although Smad3 requires the presence of FoxL2 sites to induce mouse FSHbeta, even through its consensus Smad-binding element; the human promoter, which is induced by activin independently of Smad3, also requires FoxL2 sites for its induction by activin; thus the actions of FoxL2 are not exclusively through interactions with the Smad pathway. Thus, FoxL2 plays a key role in activin induction of the FSHbeta gene, by binding to sites conserved across multiple species.

TGF-beta inhibits CYP17 transcription in H295R cells acting via activin receptor-like kinase 5.

OBJECTIVE: Transforming growth factor beta (TGF-beta) is a potent inhibitor of 17alpha-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-beta in adrenocortical cells. METHODS: H295R cells were culture and incubated with TGF-beta, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TbetaRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. RESULTS: TGF-beta significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-beta on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-beta on CYP17 expression. CONCLUSION: We concluded that the TGF-beta-dependent decrease of 17alpha-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.

TGF-beta signals regulate axonal development through distinct Smad-independent mechanisms.

Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Here I show that Transforming growth factor beta (TGFbeta) signals regulate axon growth. In most contexts, TGFbeta signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFbeta responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFbeta receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFbeta receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo.

Activin A: autocrine regulator of kidney development and repair.

The research described in this review suggests a novel and important role for activin A in the developmental and repair processes of the kidney (Table 1). The results obtained in these studies indicate that activin A is a negative regulator of kidney development and plays an essential part in kidney diseases, such as acute renal failure or renal fibrosis. It is also possible that activin A is a key player in the pathophysiological processes of other kidney diseases, such as congenital urogenital abnormalities, renal cystic disease and renal cell carcinoma. Activin A is thus a potential target for therapeutic interventions in kidney diseases. To address this issue, more detailed analysis on the regulation of activin production, modulation of activin activity and activin target genes is required.

Signal transduction pathway through activin receptors as a therapeutic target of musculoskeletal diseases and cancer.

Activin, myostatin and other members of the TGF-beta superfamily signal through a combination of type II and type I receptors, both of which are transmembrane serine/threonine kinases. Activin type II receptors, ActRIIA and ActRIIB, are primary ligand binding receptors for activins, nodal, myostatin and GDF11. ActRIIs also bind a subset of bone morphogenetic proteins (BMPs). Type I receptors that form complexes with ActRIIs are dependent on ligands. In the case of activins and nodal, activin receptor-like kinases 4 and 7 (ALK4 and ALK7) are the authentic type I receptors. Myostatin and GDF11 utilize ALK5, although ALK4 could also be activated by these growth factors. ALK4, 5 and 7 are structurally and functionally similar and activate receptor-regulated Smads for TGF-beta, Smad2 and 3. BMPs signal through a combination of three type II receptors, BMPRII, ActRIIA, and ActRIIB and four type I receptors, ALK1, 2, 3, and 6. BMPs activate BMP-specific Smads, Smad1, 5 and 8. Smad proteins undergo multimerization with co-mediator Smad, Smad4, and translocated into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. The signal transduction pathway through activin type II receptors, ActRIIA and ActRIIB, with type I receptors is involved in various human diseases. In this review, we discuss the role of signaling through activin receptors as therapeutic targets of intractable neuromuscular diseases, endocrine disorders and cancers.

Pioglitazone induces apoptosis in human vascular smooth muscle cells from diabetic patients involving the transforming growth factor-beta/activin receptor-like kinase-4/5/7/Smad2 signaling pathway.

Alterations in vascular wall remodeling are a typical complication in type 2 diabetes mellitus due to an imbalance between cell proliferation and apoptosis. In this context, we have previously shown that vascular smooth muscle cells (VSMC) from diabetic patients were resistant to induced apoptosis. Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic effects on type 2 diabetes. Here, we aimed to study whether pioglitazone was able to induce apoptosis in VSMC from diabetic patients (DP) and, if so, whether the transforming growth factor (TGF)-beta1/Smad-2 pathway was involved. We isolated human internal mammary artery VSMC from patients who had undergone coronary-artery bypass graft. Pioglitazone (100 microM) induced apoptosis in human VSMC from diabetic and nondiabetic patients (NDP), analyzed by DNA fragmentation and by degradation of Bcl-2, in high-glucose-containing medium (15 and 25 mM). This apoptotic effect was inhibited by the activin receptor-like kinase-4/5/7/Smad2 inhibitor 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (SB-431542), denoting that the TGF-beta1/Smad-2 pathway was involved. Pioglitazone rapidly increased the extracellular TGF-beta1 levels and concomitantly induced phosphorylation of Smad2 in VSMC from DP and NDP. Thus, we demonstrated that pioglitazone induced apoptosis in human VSMC from DP, which are strongly resistant to the induced apoptosis. This effect of pioglitazone might contribute in the treatment of alterations of vascular remodeling in type 2 diabetes mellitus.

The activin axis in liver biology and disease.

Activins are a closely related subgroup within the TGFbeta superfamily of growth and differentiation factors. They consist of two disulfide-linked beta subunits. Four mammalian activin beta subunits termed beta(A), beta(B), beta(C), and beta(E), respectively, have been identified. Activin A, the homodimer of two beta(A) subunits, has important regulatory functions in reproductive biology, embryonic development, inflammation, and tissue repair. Several intra- and extracellular antagonists, including the activin-binding proteins follistatin and follistatin-related protein, serve to fine-tune activin A activity. In the liver there is compelling evidence that activin A is involved in the regulation of cell number by inhibition of hepatocyte replication and induction of apoptosis. In addition, activin A stimulates extracellular matrix production in hepatic stellate cells and tubulogenesis of sinusoidal endothelial cells, and thus contributes to restoration of tissue architecture during liver regeneration. Accumulating evidence from animal models and from patient data suggests that deregulation of activin A signaling contributes to pathologic conditions such as hepatic inflammation and fibrosis, acute liver failure, and development of liver cancer. Increased production of activin A was suggested to be a contributing factor to impaired hepatocyte regeneration in acute liver failure and to overproduction of extracellular matrix in liver fibrosis. Recent evidence suggests that escape of (pre)neoplastic hepatocytes from growth control by activin A through overexpression of follistatin and reduced activin production contributes to hepatocarcinogenesis. The role of the activin subunits beta(C) and beta(E), which are both highly expressed in hepatocytes, is still quite incompletely understood. Down-regulation in liver tumors and a growth inhibitory function similar to that of beta(A) has been shown for beta(E). Contradictory results with regard to cell proliferation have been reported for beta(C). The profound involvement of the activin axis in liver biology and in the pathogenesis of severe hepatic diseases suggests activin as potential target for therapeutic interventions.

Activin receptor-like kinases: structure, function and clinical implications.

The transforming growth factor-beta (TGF-beta) superfamily regulates a multitude of cellular processes from fertilization to adulthood in vertebrates. Signaling by the TGF-beta superfamily occurs via formation of heteromeric complexes consisting of type I and type II receptors. The type I receptors, referred to as activin receptor-like kinases (ALK), lie at the epicenter of the signaling cascade as they transduce TGF-beta signals to intracellular regulators of transcription known as Smad proteins. Currently, seven ALKs have been identified in mammals. Structurally, ALKs possess an extracellular binding domain, a transmembrane domain, a GS domain that serves as the site of activation by type II receptors, and a kinase domain that activates downstream signaling molecules. ALKs mediate the effect of TGF-beta superfamily on a variety of cellular processes such as proliferation, differentiation, apoptosis, adhesion and migration, and therefore play important roles in many biological processes. Some ALKs have been implicated in several disorders, including tumorigenesis, hemorrhagic telangiectasia (HHT), immune and renal diseases, and skeletal malfunctions, suggesting that these receptors can be used as drug targets.

Smad7 and protein phosphatase 1alpha are critical determinants in the duration of TGF-beta/ALK1 signaling in endothelial cells.

BACKGROUND: In endothelial cells (EC), transforming growth factor-beta (TGF-beta) can bind to and transduce signals through ALK1 and ALK5. The TGF-beta/ALK5 and TGF-beta/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-beta signaling is an important specificity determinant for signaling responses. TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently. RESULTS: The temporal activation of TGF-beta-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-beta/ALK1 signaling pathway. Smad7 and protein phosphatase 1alpha (PP1alpha) mRNA expression levels were found to be specifically upregulated by TGF-beta/ALK1. Ectopic expression of Smad7 or PP1alpha potently inhibited TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1alpha enhanced TGF-beta/ALK1-induced signaling responses. PP1alpha interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor. CONCLUSION: Our results suggest that upon its induction by the TGF-beta/ALK1 pathway, Smad7 may recruit PP1alpha to ALK1, and thereby control TGF-beta/ALK1-induced Smad1/5 phosphorylation.