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T K Sampath

Publications and source records attributed to T K Sampath.

At least 19 recordsLinked to original sources
PubMed2000

Expression of bone morphogenetic protein receptors type-IA, -IB and -II correlates with tumor grade in human prostate cancer tissues.

Bone morphogenetic proteins (BMPs) are potential regulators of prostate cancer cell growth and metastasis that signal through an interaction with BMP membrane receptors (BMPRs) type I and type II. In the present study, Western blot and immunohistochemical analysis of BMPRs were carried out in benign and malignant human prostate tissues to explain the loss of BMP response in human prostate cancer cells. The results demonstrated that the benign prostate specimens expressed high levels of all three BMPRs. In normal prostate, BMPRs were localized predominantly to epithelial cells. Among prostate cancer specimens, well-differentiated cancers were positive for the expression of BMPR-II, BMPR-IA, and BMPR-IB, for the most part. In contrast, only 1 of 10 poorly differentiated prostate cancer cases was positive for each of the three BMPRs (P < 0.005 for all three receptors). Taken together, these results indicate that human prostate cancer cells frequently exhibit loss of expression of BMPRs and suggest that loss of BMPRs may play an important role during the progression of prostate cancer.

Blotting, Western↗
PubMed2000

Smad6 is a Smad1/5-induced smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter.

Smad6 is an inhibitory Smad that is induced by bone morphogenetic proteins (BMPs) and interferes with BMP signaling. We have isolated the mouse Smad6 promoter and identified the regions responsible for transcriptional activation by BMPs. The proximal BMP-responsive element (PBE) in the Smad6 promoter is important for the transcriptional activation by BMPs and contains a 28-base pair GC-rich sequence including four overlapping copies of the GCCGnCGC-like motif, which is a binding site for Drosophila Mad and Medea. We generated a luciferase reporter construct (3GC2-Lux) containing three repeats of the GC-rich sequence derived from the PBE. BMPs and BMP receptors induced transcriptional activation of 3GC2-Lux in various cell types, and this activation was enhanced by cotransfection of BMP-responsive Smads, i.e. Smad1 or Smad5. Moreover, direct DNA binding of BMP-responsive Smads and common-partner Smad4 to the GC-rich sequence of PBE was observed. These results indicate that the expression of Smad6 is regulated by the effects of BMP-activated Smad1/5 on the Smad6 promoter.

Animals↗
PubMed2000

Bone morphogenetic protein-7 (osteogenic protein-1) inhibits smooth muscle cell proliferation and stimulates the expression of markers that are characteristic of SMC phenotype in vitro.

Vascular proliferative disorders are characterized by migration and proliferation of vascular smooth muscle cells (SMCs), loss of expression of SMC phenotype, and enhanced extracellular matrix synthesis (e.g., type I collagen). We report here that bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor-beta (TGF-beta) superfamily, is capable of inhibiting both serum-stimulated and growth factor-induced (platelet-derived growth factor [PDGF-BB] and TGF-beta1) cell growth as measured by (3)H-thymidine uptake into DNA synthesis and cell number in primary human aortic smooth muscle (HASM) cell cultures. Concomitantly, addition of BMP-7 stimulates the expression of SMC-specific markers, namely alpha-actin and heavy chain myosin as examined by RT-PCR and Northern blot analyses. The collagen type III/I ratio that becomes lower with the transdifferentiation of SMCs into myofibroblasts is also maintained in BMP-7-treated cultures as compared to untreated controls. Studies on the mechanism of action indicate that BMP-7 treatment inhibits cyclin-dependent kinase 2 (cdk-2) that was stimulated during PDGF-BB-induced proliferation of SMCs and upregulates the expression of the inhibitory Smad, Smad6, which was shown to inhibit TGF-beta superfamily signaling. These results collectively suggest that BMP-7 maintains the expression of vascular SMC phenotype and may prevent vascular proliferative disorders, thus potentially acting as a palliative after damage to the vascular integrity.

Actins↗
PubMed2000

Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.

Actins↗
PubMed1999

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Activin Receptors↗
PubMed1999

Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation.

Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-(&bgr;) superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.

3T3 Cells↗
PubMed1998

Identification of a novel bone morphogenetic protein-responsive gene that may function as a noncoding RNA.

Bone morphogenetic proteins (BMPs)/osteogenic proteins (OPs), members of the transforming growth factor-beta superfamily, have a wide variety of effects on many cell types including osteoblasts and chondroblasts, and play critical roles in embryonic development. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. Signaling by these receptors is mediated by the recently identified Smad proteins. Despite the rapid progress in understanding of the signaling mechanism downstream of BMP receptors, the target genes of BMPs are poorly understood in mammals. Here we identified a novel gene, termed BMP/OP-responsive gene (BORG), in C2C12 mouse myoblast cell line which trans-differentiates into osteoblastic cells in response to BMPs. Expression of BORG was dramatically induced in C2C12 cells by the treatment with BMP-2 or OP-1 within 2 h and peaked at 12-24 h, whereas transforming growth factor-beta had a minimal effect. BMP-dependent expression of BORG was also detected in other cell types which are known to respond to BMPs, suggesting that BORG is a common target gene of BMPs. Cloning and sequence analysis of BORG cDNA and the genomic clones revealed that, unexpectedly, the transcript of BORG lacks any extensive open reading frames and contains a cluster of multiple interspersed repetitive sequences in its middle part. The unusual structural features suggested that BORG may function as a noncoding RNA, although it is spliced and polyadenylated as authentic protein-coding mRNAs. Together with the observation that transfection of antisense oligonucleotides of BORG partially inhibited BMP-induced differentiation in C2C12 cells, it is possible that a new class of RNA molecules may have certain roles in the differentiation process induced by BMPs.

Animals↗
PubMed1998

Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.

Acute Kidney Injury↗
PubMed1998

Induction of Smad6 mRNA by bone morphogenetic proteins.

Members of the transforming growth factor-beta (TGF-beta) superfamily transduce signals via Smad proteins. Smad2 and Smad3 mediate TGF-beta signaling, whereas Smad1 and Smad5 transduce bone morphogenetic protein (BMP) signals. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 are recently identified members in the Smad family; they inhibit the signaling activity of the other Smad proteins. Here we show that expression of the Smad6 mRNA is dramatically induced by BMP-2 or osteogenic protein-1 (OP-1)/BMP-7 in various cells. BMP-2 induced expression of Smad7 in one cell type, although much less potently than that of Smad6. Smad6 message was induced by TGF-beta 1 in TGF-beta 1-responsive Mv1Lu cells, but the induction was transient in contrast to the induction by BMPs. These results indicate that Smad6 may form a feedback loop to regulate the signaling activity of BMPs.

Animals↗
PubMed1998

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Animals↗
PubMed1997

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid.

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.

Alkaline Phosphatase↗
PubMed1997

Cooperation of BMP7 and SHH in the induction of forebrain ventral midline cells by prechordal mesoderm.

Ventral midline cells at different rostrocaudal levels of the central nervous system exhibit distinct properties but share the ability to pattern the dorsoventral axis of the neural tube. We show here that ventral midline cells acquire distinct identities in response to the different signaling activities of underlying mesoderm. Signals from prechordal mesoderm control the differentiation of rostral diencephalic ventral midline cells, whereas notochord induces floor plate cells caudally. Sonic hedgehog (SHH) is expressed throughout axial mesoderm and is required for the induction of both rostral diencephalic ventral midline cells and floor plate. However, prechordal mesoderm also expresses BMP7 whose function is required coordinately with SHH to induce rostral diencephalic ventral midline cells. BMP7 acts directly on neural cells, modifying their response to SHH so that they differentiate into rostral diencephalic ventral midline cells rather than floor plate cells. Our results suggest a model whereby axial mesoderm both induces the differentiation of overlying neural cells and controls the rostrocaudal character of the ventral midline of the neural tube.

Animals↗
PubMed1997

Effects of recombinant human osteogenic protein 1 on the production of proteoglycan, prostaglandin E2, and interleukin-1 receptor antagonist by human articular chondrocytes cultured in the presence of interleukin-1beta.

OBJECTIVE: Recombinant human osteogenic protein 1 (OP-1) is an effective stimulator of human cartilage 35S-proteoglycan synthesis. The present study was conducted to determine whether stimulation of human articular chondrocytes with OP-1 can help overcome interleukin-1beta (IL-1beta)-induced suppression of 35S-proteoglycan synthesis. METHODS: Human articular chondrocytes in alginate beads were maintained for 3 days in the absence (control) or presence of IL-1beta at 0.1-100 pg/ml with or without OP-1 at 50 ng/ml, in medium containing 10% fetal bovine serum (FBS). Incorporation of 35S-sulfate into proteoglycans was quantified during the last 4 hours of culture and reported as counts per minute per microg DNA. Release of interleukin-1 receptor antagonist (IL-1Ra) and prostaglandin E2 into the medium was monitored by immunoassay. RESULTS: IL-1beta at 10 pg/ml caused a 60% decrease in 35S-proteoglycan synthesis. This could be blocked by including 500 ng/ml IL-1Ra in the medium. The presence of 50 ng/ml OP-1 in the IL-1beta-containing medium was effective in restoring 35S-proteoglycan synthesis to the level of that found in cultures not treated with IL-1beta. The restorative effects of OP-1 and IL-1Ra were cumulative. The rate of release of prostaglandin E2 and IL-1Ra into the medium was not affected by the presence of OP-1. CONCLUSION: Treatment of human articular chondrocytes with OP-1 cultured in the presence of FBS is effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-1beta.

Adolescent↗
PubMed1997

Osteogenic protein-1 stimulates mRNA levels of BMP-6 and decreases mRNA levels of BMP-2 and -4 in human osteosarcoma cells.

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro.

Blotting, Northern↗
PubMed1997

Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum.

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Activin Receptors↗
PubMed1997

Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in human osteosarcoma cells.

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3-36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or beta-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1-24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

Actins↗
PubMed1997

Osteogenic protein-1 up-regulation of the collagen X promoter activity is mediated by a MEF-2-like sequence and requires an adjacent AP-1 sequence.

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (-310/-278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-beta2 or activin, which up-regulates the expression of a transforming growth factor-beta-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (-294/-285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (-310/-298, TTAAAAATAAAAA) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2-like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.

Activins↗