KLF10 is upregulated in osteoarthritis and inhibits chondrocyte proliferation and migration by upregulating Acvr1 and suppressing inhbb expression.

BACKGROUD: Osteoarthritis (OA) is a common disease caused by chondrocyte dysfunction. KLF10 is a member of the Sp1-like transcription factor family that is involved in regulating osteoblasts, but its expression and regulatory mechanism(s) in chondrocytes are unclear. In the present study, we aimed to investigate the regulatory role of KLF10 on the pathological process of OA. METHODS: KLF10 expression in the cartilaginous tissue of patients with osteoarthritis (OA) was evaluated by immunohistochemistry (IHC). Next, we generated an OA mouse model, and the histological changes in cartilage tissue were verified using H&E staining, Safranin O-Fast Green staining, and IHC assays. KLF10 expression in the articular chondrocytes of OA mice was determined by qRT-PCR and Western blotting. To investigate the role of KLF10 in regulating cell proliferation and migration, a KLF10 overexpression plasmid was constructed and transfected into primary mouse chondrocytes. Subsequently, we used RNA sequencing (RNA-seq) to screen differentially expressed genes in chondrocytes with or without KLF10 overexpression. qRT-PCR was used for verification purposes. RESULTS: We found that KLF10 was upregulated in the cartilaginous tissue of patients with OA as well as in cartilaginous tissue of the OA mouse model. KLF10 overexpression inhibited the proliferation and migration of chondrocytes. Furthermore, RNA sequencing results identified increased expression of Acvr1 and decreased expression of Inhbb in cells overexpressing KLF10. Changes in mRNA expression of Acvr1 and Inhbb were confirmed by qRT-PCR. CONCLUSIONS: KLF10 inhibits chondrocyte proliferation and migration by regulating the expression of Acvr1 and Inhbb in both human and mouse OA. These data suggest that KLF10 may be a potential therapeutic target for the treatment of OA.

HLA-B27 alters BMP/TGFß signalling in Drosophila, revealing putative pathogenic mechanism for spondyloarthritis.

OBJECTIVES: The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. METHODS: We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human ß2-microglobulin (hß2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA. RESULTS: Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hß2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor ß (TGFß). CONCLUSIONS: Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFß/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.

Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFß) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n = 5) and vehicle treated AlK-1-cKO mice (cKO-CON; n = 5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n = 15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p < 0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFß-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFß1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of ß-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of AlK-1 in cardiac remodeling independent of systemic AVMs are required.

BMP signaling pathways affect differently migration and invasion of esophageal squamous cancer cells.

Bone morphogenetic proteins (BMPs) are broadly involved in normal embryo development and abnormal pathological process such as cancer. The complexity and diversity of BMPs and their signaling pathways impose quite different or even conflicting effects on clinical traits of tumors. The aim of the present study was to investigate whether different BMPs, including BMP2, BMP4, BMP6 and BMP7, influence esophageal squamous cancer cell (ESCC) growth, invasion and metastasis. BMP6 and type I receptor ALK2 and type II receptor BMPRII, ActRIIA and ActRIIB were expressed in all ESCC cell lines. In addition, adenovirus-mediated BMP overexpression did not affect ECA-109 cell growth. BMP6/7 overexpression increased ECA-109 cell invasion and metastasis, activated SMAD1/5/8 signal pathway and induced downstream gene ID1 expression. While BMP2/4 overexpression reduced ECA-109 cell invasion and metastasis and obviously promoted ERK1/2, P-38 and JNK activation with weak SMAD1/5/8 phosphorylation. When BMP6/7 favorite type I receptor ALK2 or type II receptor BMPRII was interfered with by dominant-negative mutation, BMP6/7-induced invasion and metastasis augmentation disappeared. Further investigation on clinical ESCC samples and non-tumorous adjacent tissue found that tumors with triple-positive BMP6, ALK2 and BMPRII had deeper growth than tumors with only BMP6 expression. These results suggested that different BMPs distinctly affected esophageal squamous cancer cell invasion and metastasis by employing different signal pathways.

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response.

Endoglin is an auxiliary receptor for members of the TGF-ß superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-ß receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng(fl/fl)LysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng(fl/fl)LysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-ß1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.

BMP9 Induces Cord Blood-Derived Endothelial Progenitor Cell Differentiation and Ischemic Neovascularization via ALK1.

OBJECTIVE: Modulating endothelial progenitor cells (EPCs) is essential for therapeutic angiogenesis, and thus various clinical trials involving EPCs are ongoing. However, the identification of environmental conditions and development of optimal methods are required to accelerate EPC-driven vasculogenesis. APPROACH AND RESULTS: We evaluated gene expression profiles of cord blood-derived EPCs and endothelial cells to identify the key factors in EPC→endothelial cell differentiation and to show that transforming growth factor-ß family members contribute to EPC differentiation. The expression levels of activin receptor-like kinase 1 (ALK1) and its high-affinity ligand, bone morphogenetic protein 9 (BMP9) were markedly changed in EPC→endothelial cell differentiation. Interestingly, BMP9 induced EPC→endothelial cell differentiation and EPC incorporation into vessel-like structures by acting on ALK1 expressed on EPCs in vitro. BMP9 also induced neovascularization in mice with hindlimb ischemia by increasing vessel formation and the incorporation of EPCs into vessels. Conversely, neovascularization was impaired when ALK1 signaling was blocked. Furthermore, EPCs exposed to either short- or long-term BMP9 stimulation demonstrated these functions in EPC-mediated neovascularization. CONCLUSIONS: Collectively, our results indicated that BMP9/ALK1 augmented vasculogenesis and angiogenesis, and thereby enhanced neovascularization. Thus, we suggest that BMP9/ALK1 may improve the efficacy of EPC-based therapies for treating ischemic diseases.

PU.1-Regulated Long Noncoding RNA lnc-MC Controls Human Monocyte/Macrophage Differentiation through Interaction with MicroRNA 199a-5p.

Long noncoding RNAs (lncRNAs) are emerging as important regulators in mammalian development, but little is known about their roles in monocyte/macrophage differentiation. Here we identified a long noncoding monocytic RNA (lnc-MC) that exhibits increased expression during monocyte/macrophage differentiation of THP-1 and HL-60 cells as well as CD34(+) hematopoietic stem/progenitor cells (HSPCs) and is transcriptionally activated by PU.1. Gain- and loss-of-function assays demonstrate that lnc-MC promotes monocyte/macrophage differentiation of THP-1 cells and CD34(+) HSPCs. Mechanistic investigation reveals that lnc-MC acts as a competing endogenous RNA to sequester microRNA 199a-5p (miR-199a-5p) and alleviate repression on the expression of activin A receptor type 1B (ACVR1B), an important regulator of monocyte/macrophage differentiation. We also noted a repressive effect of miR-199a-5p on lnc-MC expression and function, but PU.1-dominant downregulation of miR-199a-5p weakens the role of miR-199a-5p in the reciprocal regulation between miR-199a-5p and lnc-MC. Altogether, our work demonstrates that two PU.1-regulated noncoding RNAs, lnc-MC and miR-199a-5p, have opposing roles in monocyte/macrophage differentiation and that lnc-MC facilitates the differentiation process, enhancing the effect of PU.1, by soaking up miR-199a-5p and releasing ACVR1B expression. Thus, we reveal a novel regulatory mechanism, comprising PU.1, lnc-MC, miR-199a-5p, and ACVR1B, in monocyte/macrophage differentiation.

Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells.

Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also suggested that mutual enhancement of melatonin and activin receptor signaling is involved in the induction of aldosterone output by adrenocortical cells.

Mutant activin-like kinase 2 in fibrodysplasia ossificans progressiva are activated via T203 by BMP type II receptors.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by progressive heterotopic ossification in soft tissues, such as the skeletal muscles. FOP has been shown to be caused by gain-of-function mutations in activin receptor-like kinase (ALK)-2, which is a type I receptor for bone morphogenetic proteins (BMPs). In the present study, we examined the molecular mechanisms that underlie the activation of intracellular signaling by mutant ALK2. Mutant ALK2 from FOP patients enhanced the activation of intracellular signaling by type II BMP receptors, such as BMPR-II and activin receptor, type II B, whereas that from heart disease patients did not. This enhancement was dependent on the kinase activity of the type II receptors. Substitution mutations at all nine serine and threonine residues in the ALK2 glycine- and serine-rich domain simultaneously inhibited this enhancement by the type II receptors. Of the nine serine and threonine residues in ALK2, T203 was found to be critical for the enhancement by type II receptors. The T203 residue was conserved in all of the BMP type I receptors, and these residues were essential for intracellular signal transduction in response to ligand stimulation. The phosphorylation levels of the mutant ALK2 related to FOP were higher than those of wild-type ALK2 and were further increased by the presence of type II receptors. The phosphorylation levels of ALK2 were greatly reduced in mutants carrying a mutation at T203, even in the presence of type II receptors. These findings suggest that the mutant ALK2 related to FOP is enhanced by BMP type II receptors via the T203-regulated phosphorylation of ALK2.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

Increased decidual mRNA expression levels of candidate maternal pre-eclampsia susceptibility genes are associated with clinical severity.

INTRODUCTION: Pre-eclampsia (PE) has a familial association, with daughters of women who had PE during pregnancy having more than twice the risk of developing PE themselves. Through genome-wide linkage and genetic association studies in PE-affected families and large population samples, we previously identified the following as positional candidate maternal susceptibility genes for PE; ACVR1, INHA, INHBB, ERAP1, ERAP2, LNPEP, COL4A1 and COL4A2. The aims of this study were to determine mRNA expression levels of previously identified candidate maternal pre-eclampsia susceptibility genes from normotensive and severe PE (SPE) pregnancies and correlate mRNA expression levels with the clinical severity of SPE. METHODS: Third trimester decidual tissues were collected from both normotensive (n = 21) and SPE pregnancies (n = 24) and mRNA expression levels were determined by real-time PCR. Gene expression was then correlated with several parameters of clinical severity in SPE. Statistical significance was determined by Mann-Whitney U test and Spearman's Correlation. RESULTS: The data demonstrate significantly increased decidual mRNA expression levels of ACVR1, INHBB, ERAP1, ERAP2, LNPEP, COL4A1 and COL4A2 in SPE (p < 0.05). Increased mRNA expression levels of several genes - INHA, INHBB, COL4A1 and COL4A2 were correlated with earlier onset of PE and earlier delivery of the fetus (p < 0.05). CONCLUSION: These results suggest altered expression of maternal susceptibility genes may play roles in PE development and the course of disease severity.

Expression of activin receptor-like kinase 7 in adipose tissues.

The tissue distribution of activin receptor-like kinase 7 (Alk7) expression, the signaling ability of Alk7 variants, and Alk7 expression in response to ß3-adrenergic receptor activation were examined. Expression levels of Alk7 varied greatly among tissues but were highest in white adipose tissue and brown adipose tissue. In addition to full-length Alk7 (Alk7-v1), Alk7-v3, an Alk7 variant, was expressed in adipose tissues, brain, and ovary. Nodal transmits signals via Alk7 in cooperation with its coreceptor, Cripto. Evaluation of the ability of Alk7 variants to confer Nodal signaling using luciferase-based reporter assays showed that Alk7-v3 does not transmit Nodal-Cripto-mediated signals. Expression of Alk7 was down-regulated in brown but not in white adipose tissue treated with CL316,243, a ß3-adrenergic receptor agonist. These results suggest involvement of Alk7 in modulation of metabolism in the adipose tissues in response to ß3-adrenergic receptor activation.

MicroRNA miR-98 inhibits tumor angiogenesis and invasion by targeting activin receptor-like kinase-4 and matrix metalloproteinase-11.

Angiogenesis and invasion are essential processes for solid tumor growth and dissemination. The tumor development process can be dependent on the activation of a series of signaling pathways, including growth factor-activated pathways. MicroRNAs have been shown to be critical for tumorigenesis, but their roles in cancer angiogenesis, invasion and other signaling pathways important for tumor development are still unclear in the context of tumor biology. We investigated the role of microRNA miR-98 in regulating tumor growth, invasion, and angiogenesis using a highly aggressive breast cancer model in vitro and in vitro. We found that the expression of miR-98 inhibited breast cancer cell proliferation, survival, tumor growth, invasion, and angiogenesis. Conversely, inhibition of endogenous miR-98 promoted cell proliferation, survival, tumor growth, invasion, and angiogenesis. It appeared that miR-98 inhibited angiogenesis by modulating endothelial cell activities including cell spreading, cell invasion and tubule formation. Interestingly, miR-98 reduced the expression of ALK4 and MMP11, both of which were potential targets of miR-98. Transfection of an anti-miR-98 construct increased the expression of both targets. We confirmed that mir-98 targeted the 3'-untranslated regions of ALK4 and MMP11. Finally, ALK4- and MMP11-specific siRNAs inhibited breast cancer cell proliferation, survival, and angiogenesis. Rescue experiments with ALK4 and MMP11 constructs reversed the anti-proliferative, anti-invasive and anti-angiogenic effects of miR-98. Our findings define a regulatory role of miR-98 in tumor angiogenesis and invasion through repressed ALK4 and MMP11 expression.

Expression of myostatin, myostatin receptors and follistatin in diabetic rats submitted to exercise.

Myostatin (MSTN) has been implicated in metabolic adaptation to physiological stimuli, such as physical exercise, which is linked to improved glucose homeostasis. The aim of the present study was to evaluate the influence of exercise on the expression of MSTN, MSTN receptors (ActRIIB and ALK4) and follistatin (FS) in the muscle and fat of streptozotocin-induced diabetic rats. Control and diabetic rats were randomly assigned to a swimming training group (EC and ED, respectively) and a sedentary group (SC and SD, respectively). Exercising animals swam for 45 min at 0900 and 1700 hours, 5 day/week, for 4 weeks. The mRNA expression of MSTN, ActRIIB, ALK4 and FS mRNA was quantified by real-time reverse transcription-polymerase chain reaction. Expression of MSTN and FS mRNA increased in the muscle and subcutaneous fat of SD compared with SC rats. Expression of ActRIIB mRNA was increased in the muscle, mesenteric fat and brown adipose tissue (BAT) of SD compared with SC rats, whereas ALK4 mRNA expression was only increased in the BAT of SD compared with SC rats. After training, MSTN and ActRIIB expression was lower in the BAT of EC compared with SC rats. Expression of MSTN mRNA increased in the mesenteric fat of ED compared with SD rats, whereas FS mRNA expression decreased in the muscle, mesenteric and subcutaneous fat and BAT. Lower ALK4 mRNA expression was noted in the BAT of ED compared with SD rats. These results indicate that MSTN, its receptors and FS expression change in both the muscle and fat of diabetic rats and that the expression of these factors can be modulated by exercise in diabetes.

The expression and potential function of bone morphogenetic proteins 2 and 4 in bovine trophectoderm.

BACKGROUND: Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for BMP2 and 4, an innate inhibitor noggin (NOG), and BMP2/4 receptors (BMPRII, ACVR1, BMPR1A, BMPR1B), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression. METHODS: RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated. RESULTS: Transcripts for BMP2 and 4 were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for NOG were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced IFNT mRNA abundance but had no effect on CSH1 mRNA abundance in CT1 cells. CONCLUSIONS: The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.

BMP-11 and myostatin support undifferentiated growth of human embryonic stem cells in feeder-free cultures.

BMP-11/GDF-11 and Myostatin/GDF-8 are both members of the TGF-beta superfamily that can activate SMAD2/3 phosphorylation via the type I receptors ALK4, ALK5, or ALK7. We tested the ability of BMP-11 and Myostatin to promote self-renewal of human embryonic stem cells (hESC) under feeder-free and serum-free culture conditions in short term (1 week) and medium term cultures (10 weeks). We show that hESC cultured in serum-free medium supplemented with either 20 ng/mL Myostatin or 20 ng/mL BMP-11 maintain the colony and cellular morphology of undifferentiated hESC, maintain POU5f1, NANOG, TRA-1-60, and SSEA4 expression, and display increased SMAD2/3 phosphorylation, similar to hESC cultured in mouse embryonic fibroblast feeder-CM or 20 ng/mL Activin-A. The type I TGF-beta receptor inhibitor SB431542 totally inhibited the maintenance activity of both Myostatin or BMP-11 supplemented medium. Our data show that members of the TGF-beta superfamily, other than Activin-A and GDF3, are able to maintain hES cells in an undifferentiated state under feeder free conditions.

Cripto localizes Nodal at the limiting membrane of early endosomes.

Cripto is a glycosylphosphatidylinositol (GPI)-anchored co-receptor of Nodal and several other transforming growth factor-beta (TGF-beta) family ligands. It contains an epidermal growth factor (EGF)-like motif and a Cripto-FRL1-Cryptic (CFC) domain, which are conserved in a family of EGF-CFC proteins. The EGF domain is thought to recruit Nodal, whereas the CFC domain mediates binding to activin receptor-like kinase 4 (ALK4). We found that the EGF-like motif of Cripto was not essential for its binding to Nodal. However, through residues phenylalanine 78 and glycine 71, Cripto enriched Nodal at the limiting membrane of early endosomes. Similarly, residues in the CFC domain that mediate binding of Cripto to ALK4 were required to attenuate sequestration of Nodal in the endosomal lumen. Thus, we propose that Cripto stimulates Nodal activity by localizing it at the interface of endosomes with cytoplasmic effectors. To our knowledge, Cripto is the first GPI-anchored protein shown to control intraendosomal sorting of its associated cargo.

Expression of BMP7 is associated with resistance to diabetic stress: comparison among mouse salivary glands.

We determined mRNA levels of bone morphogenetic protein 7 (BMP7), a growth and differentiation factor belonging to the transforming growth factor-beta superfamily, in the salivary glands of mice with streptozotocin (200 mg/kg, i.p.)-induced diabetes. We also examined the effects of BMP7 on secretion of saliva and degenerative change in salivary glands in diabetic mice. In normal mice, BMP7 mRNA levels were high in the submandibular gland and low in the parotid gland, while in diabetic mice, levels were significantly decreased in the parotid gland, but not in the submandibular gland. No significant difference was observed in mRNA levels of BMP receptors between normal and diabetic mice. In diabetic mice, pilocarpine (4 mg/kg, i.p.)-stimulated salivary secretion showed a remarkable decrease in both parotid and submandibular gland, although degree of reduction was smaller in the latter. Notable degeneration with vacuolation and atrophy was also found in parotid gland, whereas degeneration of submandibular gland was slight. Administration of BMP7 (50 and 100 microg/kg, i.v.) in diabetic mice induced a significant increase in salivary secretion, with rate of recovery higher in parotid gland than in submandibular gland. In diabetic mice, BMP7 also exhibited a powerful protective effect in degenerated salivary gland, especially in parotid gland. These results suggest that BMP7 acts to prevent diabetic damage in salivary gland, and that its cytoprotective effect is closely correlated with mRNA levels in tissue.

MicroRNA miR-24 inhibits erythropoiesis by targeting activin type I receptor ALK4.

MicroRNAs have been suggested to modulate a variety of cellular events. Here we report that miR-24 regulates erythroid differentiation by influencing the expression of human activin type I receptor ALK4 (hALK4). Ectopic expression of miR-24 reduces the mRNA and protein levels of hALK4 by targeting the 3'-untranslated region of hALK4 mRNA and interferes with activin-induced Smad2 phosphorylation and reporter expression. Furthermore, miR-24 represses the activin-mediated accumulation of hemoglobin, an erythroid differentiation marker, in erythroleukemic K562 cells and decreases erythroid colony-forming and burst-forming units of CD34+ hematopoietic progenitor cells. ALK4 expression is inversely correlated with miR-24 expression during the early stages of erythroid differentiation, and the forced expression of miR-24 leads to a delay of activin-induced maturation of hematopoietic progenitor cells in liquid culture. Thus, our findings define a regulation mode of miR-24 on erythropoiesis by impeding ALK4 expression.

Rapid closure of midgestational excisional wounds in a fetal mouse model is associated with altered transforming growth factor-beta isoform and receptor expression.

BACKGROUND: Many pediatric diseases are characterized by excessive tissue contraction. Because of a poor understanding of contraction, few therapies exist. We developed a murine fetal excisional wound model of contraction and theorize that wound closure is associated with changes in transforming growth factor-beta (TGF-beta) expression. METHODS: Pregnant FVB mice underwent hysterotomy at midgestational (E15) or late-gestational (E18) ages. Three-millimeter excisional wounds were made in fetuses and harvested at 32 hours. Real-time polymerase chain reaction was performed for TGF-beta1, TGF-beta2, TGF-beta3, TbetaR-1, and TbetaR-2 in wounds and normal skin and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by paired t test (P < .05). H&E staining of wounds was performed. RESULTS: E15 wounds (80.5% +/- 4.4%) were smaller than E18 wounds (10.4% +/- 10.5%; P < .001) at 32 hours. E15 wounds expressed higher levels of TGF-beta1 compared with normal skin (P = .001). TbetaR-2 levels were elevated in E15 and E18 wounds compared with their respective normal skin (P = .02, P = .01) and in E18 normal skin compared with E15 normal skin (P = .002). CONCLUSION: This study demonstrates that rapid midgestational wound closure in a murine model is associated with increased TGF-beta1 and TbetaR-2 expression. Elucidating the role of the TGF-beta pathways may lead to an improved understanding of wound contraction.