Coicis semen protects against focal cerebral ischemia-reperfusion injury by inhibiting oxidative stress and promoting angiogenesis via the TGFß/ALK1/Smad1/5 signaling pathway.

BACKGROUND: Ischemic stroke is a devastating disease that causes long-term disability. However, its pathogenesis is unclear, and treatments for ischemic stroke are limited. Recent studies indicate that oxidative stress is involved in the pathological progression of ischemic stroke and that angiogenesis participates in recovery from ischemic stroke. Furthermore, previous studies have shown that Coicis Semen has antioxidative and anti-inflammatory effects in a variety of diseases. In the present study, we investigated whether Coicis Semen has a protective effect against ischemic stroke and the mechanism of this protective effect. RESULTS: Coicis Semen administration significantly decreased the infarct volume and mortality and alleviated neurological deficits at 3, 7 and 14 days after MCAO. In addition, cerebral edema at 3 days poststroke was ameliorated by Coicis Semen treatment. DHE staining showed that ROS levels in the vehicle group were increased at 3 days after reperfusion and then gradually declined, but Coicis Semen treatment reduced ROS levels. The levels of GSH and SOD in the brain were increased by Coicis Semen treatment, while MDA levels were reduced. Furthermore, Coicis Semen treatment decreased the extravasation of EB dye in MCAO mouse brains and elevated expression of the tight junction proteins ZO-1 and Occludin. Double immunofluorescence staining and western blot analysis showed that the expression of angiogenesis markers and TGFß pathway-related proteins was increased by Coicis Semen administration. Consistent with the in vivo results, cytotoxicity assays showed that Coicis Semen substantially promoted HUVEC survival following OGD/RX in vitro. Additionally, though LY2109761 inhibited the activation of TGFß signaling in OGD/RX model animals, Coicis Semen cotreatment markedly reversed the downregulation of TGFß pathway-related proteins and increased VEGF levels. METHODS: Adult male wild-type C57BL/6J mice were used to develop a middle cerebral artery occlusion (MCAO) stroke model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7 and 14 after staining. In addition, changes in superoxide dismutase (SOD), GSH and malondialdehyde (MDA) levels were detected with a commercial kit. Blood-brain barrier (BBB) permeability was assessed with Evans blue (EB) dye. Western blotting was also performed to measure the levels of tight junction proteins of the BBB. Additionally, ELISA was performed to measure the level of VEGF in the brain. The colocalization of CD31, angiogenesis markers, and Smad1/5 was assessed by double immunofluorescent staining. TGFß pathway-related proteins were measured by western blotting. Furthermore, the cell viability of human umbilical vein endothelial cells (HUVECs) following oxygen-glucose deprivation/reoxygenation (OGD/RX) was measured by Cell Counting Kit (CCK)-8 assay. CONCLUSIONS: Coicis Semen treatment alleviates brain damage induced by ischemic stroke through inhibiting oxidative stress and promoting angiogenesis by activating the TGFß/ALK1 signaling pathway.

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

Reversal of Sp1 transactivation and TGFß1/SMAD1 signaling by H2S prevent nickel-induced fibroblast activation.

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200 µM) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1 mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFß1/SMAD1 signal and blocked TGFß1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFß1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.

BMP6 Downregulates GDNF Expression Through SMAD1/5 and ERK1/2 Signaling Pathways in Human Granulosa-Lutein Cells.

Bone morphogenetic protein (BMP) 6 is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line‒derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein (hGL) cells as in vitro cell models. Our results showed that BMP6 significantly downregulated the expression of GDNF in both SVOG and primary hGL cells. With dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both activin receptor kinase-like (ALK) 2 and ALK3 are involved in BMP6-induced downregulation of GDNF. In addition, BMP6 induced the phosphorylation of Sma- and Mad-related protein (SMAD)1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced downregulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced downregulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through paracrine interactions in human granulosa cells.

ALK2/ALK3-BMPR2/ACVR2A Mediate BMP2-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa-Lutein Cells.

Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-ß superfamily and plays a critical role in regulating ovarian follicle function. Currently, the role of BMP2 during cumulus expansion remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of pentraxin 3 (PTX3) expression (the major component of cumulus expansion) and the underlying mechanisms in human granulosa-lutein (hGL) cells. Both primary and immortalized hGL cells were used as research models. Our results showed that treatment with BMP2 significantly suppressed the basal and luteinizing hormone-induced upregulation of PTX3. In addition, BMP2 stimulated the phosphorylation of SMAD1/5/8, and this effect was abolished by the addition of BMP type I receptor inhibitors, dorsomorphin homolog 1, and dorsomorphin but not SB431542. Moreover, the knockdown of activin receptorlike kinase 2/3 or BMP receptor type II/activin receptor type IIB receptors completely reversed the BMP2-induced phosphorylation of SMAD1/5/8 and restored PTX3 expression. Similarly, the knockdown of SMAD4 completely reversed the suppressive effect of BMP2 on the expression of PTX3. These results improve our understanding of the molecular mechanisms of BMP2 signaling. Our findings suggest that BMP2 may be involved in the regulation of cumulus expansion during the periovulatory stage.

Activin B Induces Noncanonical SMAD1/5/8 Signaling via BMP Type I Receptors in Hepatocytes: Evidence for a Role in Hepcidin Induction by Inflammation in Male Mice.

Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation. Bone morphogenetic protein 6 (BMP6) signaling is a central regulator of hepcidin expression in the liver. Recently, the TGF-ß/BMP superfamily member activin B was implicated in hepcidin induction by inflammation via noncanonical SMAD1/5/8 signaling, but its mechanism of action and functional significance in vivo remain uncertain. Here, we show that low concentrations of activin B, but not activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2/3 signaling, and with similar or modestly reduced potency compared with BMP6. Activin B stimulates hepcidin via classical activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors activin receptor-like kinase 2 and activin receptor-like kinase 3, and SMAD5. The coreceptor hemojuvelin binds to activin B and facilitates activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling has some selectivity for hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Liver activin B mRNA expression is up-regulated in multiple mouse models of inflammation associated with increased hepcidin and hypoferremia, including lipopolysaccharide, turpentine, and heat-killed Brucella abortus models. Finally, the activin inhibitor follistatin-315 blunts hepcidin induction by lipopolysaccharide or B. abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation and support a likely functional role for activin B in hepcidin stimulation during inflammation in vivo.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

The Effect of Tumor Necrosis Factor-α at Different Concentrations on Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Tumor necrosis factor-alpha (TNF-α) has been demonstrated to have a close relationship with inflammation in the body. Although most researchers confirmed that TNF-α can have an effect on the expression of osteoblast specific genes, they had not elucidated the regulation of inflammatory factors in osteogenic gene expression during the process of bone marrow mesenchymal stem cells (BMMSCs) differentiating to osteoblast. The aim of this study was to investigate the effect of TNF-α at different concentrations on osteogenetic differentiation of BMMSCs. In this study, BMMSCs proliferation was analyzed by using cell counting kit-8 assay, cell osteogenic differentiation was evaluated by means of alkaline phosphatase activity assay and Von Koaas staining and the messenger RNA (mRNA) expression of bone morphogenetic proteins-2 (BMP-2) and drosophila mothers against decapentaplegic protein 1 (Smad1) was measured through real-time polymerase chain reaction. The results indicated that a low concentration of TNF-α at short-term promotes the osteogenetic differentiation of BMMSCs and increases the mRNA expression of BMP-2 and Smad1, but inhibits the osteogenetic differentiation of BMMSCs and the expression of BMP-2 and Smad1 at long term. In addition, regardless of a short or long time, a high concentration of TNF-α inhibits the osteogenetic differentiation of BMMSCs and the expression of Smad1, but results in a high expression of BMP-2.

Recombinant biglycan promotes bone morphogenetic protein-induced osteogenesis.

The aim of this study was to determine the effects of glutathione-S-transferase-fused recombinant biglycan (GST-BGN) on craniofacial bone regeneration. We recently demonstrated a positive effect of tissue-derived BGN on bone morphogenetic protein 2 (BMP-2) function, which is exerted likely via the BGN core protein. Here, we investigated the effects of GST-BGN lacking any posttranslational modifications on BMP-2 function in vitro and in vivo. In the C2C12 cell culture system, BMP-2-induced Smad 1/5/8 phosphorylation and alkaline phosphatase activity were both enhanced by the addition of GST-BGN. For the in vivo effect, we employed a Sprague-Dawley rat mandible defect model utilizing 1 µg (optimal) or 0.1 µg (suboptimal) of BMP-2 combined with 0, 2, 4, or 8 µg of GST-BGN. At 2 weeks post-surgery, newly formed bone was evaluated by microcomputed tomography and histologic analyses. The results revealed that the greatest amounts of bone within the defect were formed in the groups of suboptimal BMP-2 combined with 4 or 8 µg of GST-BGN. Also, bone was well organized versus that formed by the optimal dose of BMP. These results indicate that recombinant BGN is an efficient substrate to promote low-dose BMP-induced osteogenesis.

Bone morphogenetic protein 6 stimulates mineralization in human dental follicle cells without dexamethasone.

OBJECTIVE: The aim of this study is to investigate the osteogenic differentiation human dental follicle cells (hDFCs) cultured with in osteogenic induction medium (OIM) without dexamethasone (DEX), and to analyze the gene expression profile during osteogenic differentiation. METHODS: hDFCs, which isolated from dental follicle tissue from impacted third molar teeth, were cultured with OIM with or without DEX. Osteogenic differentiation of hDFCs was examined using Alkaline phosphatase activity and Arizarin red staining. Gene expression analysis was performed by Microarray and real time-PCR. RESULTS: We showed that hDFCs have the capacity to differentiate into osteogenic lineages in osteogenic induction medium lacking DEX. We also analyzed gene expression profiling of hDFCs during osteogenic differentiation. BMP6 is up-regulated in both the presence and absence of DEX. In addition, BMP6 enhances gene expression levels of DLX-5, Runx2, and Osterix, which are transcription factors associated with osteogenic differentiation. BMP6 also stimulates phosphorylation of Smad1/5/8 which are transcription factors associated with BMP signalling at protein levels. Additionally BMP6 stimulates mineralization of hDFCs monolayers examined by Arizarin red S staining. CONCLUSION: These findings suggest that hDFCs can differentiate to osteogenic lineage cells osteogenic induction medium without DEX, and BMP6 is a key gene in the osteogenic differentiation of hDFCs, and has therapeutic utility for bone regeneration and bone research.

MicroRNA-26a is strongly downregulated in melanoma and induces cell death through repression of silencer of death domains (SODD).

Melanoma is an aggressive cancer that metastasizes rapidly and is refractory to conventional chemotherapies. Identifying microRNAs (miRNAs) that are responsible for this pathogenesis is therefore a promising means of developing new therapies. We identified miR-26a through microarray and quantitative reverse-transcription-PCR (qRT-PCR) experiments as an miRNA that is strongly downregulated in melanoma cell lines as compared with primary melanocytes. Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared with a negative control in most melanoma cell lines tested. In surveying targets of miR-26a, we found that protein levels of SMAD1 (mothers against decapentaplegic homolog 1) and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic as compared with the control. The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3' untranslated region (3'UTR) of SODD (silencer of death domains). Knockdown of these proteins with small interfering RNA (siRNA) showed that SODD has an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, whereas SMAD1 may have a minor role. Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression. Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD, in particular, is a potentially useful therapeutic target.

Toxicarioside A, isolated from tropical Antiaris toxicaria, blocks endoglin/TGF-ß signaling in a bone marrow stromal cell line.

OBJECTIVE: To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells. METHODS: HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-ß were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-ß (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells. RESULTS: Compared with the not treated (0 µg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-ß expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells. CONCLUSIONS: Our results indicate that toxicarioside A can influence bone marrow stromal HS-5's function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.

Effects of recombinant dentin sialoprotein in dental pulp cells.

Dentin sialophosphoprotein (DSPP) is critical for dentin mineralization. However, the function of dentin sialoprotein (DSP), the cleaved product of DSPP, remains unclear. This study aimed to investigate the signal transduction pathways and effects of recombinant human DSP (rh-DSP) on proliferation, migration, and odontoblastic differentiation in human dental pulp cells (HDPCs). The exogenous addition of rh-DSP enhanced the proliferation and migration of HDPCs in dose- and time-dependent manners. rh-DSP markedly increased ALP activity, calcium nodule formation, and levels of odontoblastic marker mRNA. rh-DSP increased BMP-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist, noggin. Furthermore, rh-DSP phosphorylated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), Akt, and IκB-α, and induced the nuclear translocation of the NF-κB p65 subunit. Analysis of these data demonstrates a novel signaling function of rh-DSP for the promotion of growth, migration, and differentiation in HDPCS via the BMP/Smad, JNK, ERK, MAPK, and NF-κB signaling pathways, suggesting that rh-DSP may have therapeutic utility in dentin regeneration or dental pulp tissue engineering.

Levosimendan up-regulates transforming growth factor-beta and smad signaling in the aorta in the early stage of sepsis.

BACKGROUND: This prospective, controlled experimental study was planned to investigate the effects of levosimendan on transforming growth factor (TGF)-beta3 and Smad1, Smad2 and Smad3 expression in the early stages of sepsis. METHODS: Twenty-four rats were randomized into four groups: (1) sham-operated controls, (2) dobutamine group--subjected to abdominal hypertension and peritonitis-induced sepsis using cecal ligation and puncture (CLP), then treated with 10 microg x kg(-1) min(-1) intravenous (IV) dobutamine infusion, (3) levosimendan group--as in 2, then treated with levosimendan IV bolus 200 microg x kg(-1) followed by 200 microg x kq(-1) min(-1) IV infusion, and (4) a control group as in 2, with no treatment. All rats were killed 8 hours after CLP. Aorta tissue samples were analyzed by immunohistochemical staining. RESULTS: CLP caused mild interleukin (IL)-1 immunostaining in both control and dobutamine groups. Immunoreactivity of tumor necrosis factor (TNF)-alpha was mild in both sham and control groups. TGF-beta3 immunostaining was mildly increased in groups sham, control and dobutamine, whereas it was found moderate in group levosimendan. Smad1, Smad2 and Smad3 were found moderately increased only in group levosimendan. CONCLUSION: Beneficial effects of levosimendan on hemodynamics and global oxygen transport were reported in experimental and clinical trials. Besides its potency on C++ ion sensitivity, it should influence inflammatory cytokine production by diminishing TGF-beta3 and Smad1, Smad2 and Smad3 expression.

Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate.

OBJECTIVE: Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate. METHODS: Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA. RESULTS: Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor beta-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts. CONCLUSION: Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound. This study suggests that SSc patients with activated Smad1 signaling may benefit from imatinib mesylate treatment.

Upregulation of ID protein by growth and differentiation factor 5 (GDF5) through a smad-dependent and MAPK-independent pathway in HUVSMC.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

BMP-6 inhibits human bone marrow B lymphopoiesis--upregulation of Id1 and Id3.

OBJECTIVE: In mammals, factors produced by bone marrow (BM) stromal cells are instrumental in orchestrating the developmental process of B lymphocytes. Bone morphogenetic proteins (BMPs) are multifunctional cytokines previously found to regulate hematopoietic stem cells. In the present study, we have explored the role of BMP-6 in human B progenitor cells. MATERIALS AND METHODS: In vitro B lymphopoiesis of CD10(+) B progenitor cells from human BM was evaluated in the presence or absence of BMP-6 in short- or long-term coculture on MS-5 stromal cells, by tracking CFSE-labeled CD10(+) B progenitor cells or by quantification of CD19(+) cells. DNA synthesis in the pre-B cell line Nalm-6 was measured by (3)H-thymidine incorporation. BMP-6-induced phosphorylation of Smad1/5/8 was determined by Western blot analysis, whereas elevation of Id1-Id4 mRNA levels and basal BMP-6 mRNA levels were measured by real-time and conventional RT-PCR, respectively. RESULTS: By in vitro coculture of CD10(+) B progenitor cells or monoculture of Nalm-6 cells, we found that BMP-6 inhibited B lymphopoiesis by impeding cell proliferation. Furthermore, in CD10(+) B progenitors as well as in Nalm-6 cells, BMP-6 rapidly induced phosphorylation of Smad1/5/8, followed by an upregulation of Id1 and Id3 mRNA levels. Finally, we demonstrated that human bone marrow stromal cells express BMP-6 mRNA whereas B progenitor cells did not. CONCLUSIONS: We suggest that BMP-6, produced by the BM, may participate to fine-tune the balance between proliferation, apoptosis, and differentiation in human B progenitor cells during BM B lymphopoiesis.