Effect of CH-35, a novel anti-tumor colchicine analogue, on breast cancer cells overexpressing the ßIII isotype of tubulin.

The subunit protein of microtubules is tubulin, which has been the target for some of the most successful and widely used anti-tumor drugs. Most of the drugs that target tubulin bind to the ß subunit. There are many isotypes of ß-tubulin and their distributions differ among different tissues. The ßIII isotype is over-expressed in many tumors, particularly those that are aggressive, metastatic, and drug resistant. We have previously reported the design and synthesis of a series of compounds to fit the colchicine site on ßIII but not on the other isotypes. In the current study, we tested the toxicity and the anti-tumor activity of one of these compounds, CH-35, on the human breast tumor MDA-MB-231 over-expressing ßIII in a xenogeneic mouse model. We found that CH-35 was as toxic as Taxol® in vivo. Although the ßIII-over-expressing cells developed into very fast-growing tumors, CH-35 was more effective against this tumor than was Taxol. Our results suggest that CH-35 is a promising candidate for future drug development.

Long-Range Communication Network in the Type 1B Bone Morphogenetic Protein Receptor.

Protein-protein interactions are recognized as a fundamental phenomenon that is intimately associated with biological functions and thus are ideal targets for developing modulators for regulating biological functions. A challenge is to identify a site that is situated away from but functionally connected to the protein-protein interface. We employed bone morphogenetic proteins (BMPs) and their receptors as a model system to develop a strategy for identifying such a network of communication. Accordingly, using computational analyses with the COREX/BEST algorithm, we uncovered an overall pattern connecting various regions of BMPR-1B ectodomain, including the four conserved residues in the protein-protein interface. In preparation for testing the long-range effects of mutations of distal residues for future studies, we examined the extent of measurable perturbation of the four conserved residues by determination of the conformation and relative affinities of these BMPR-1B mutants for ligands BMP-2, -6, and -7 and GDF-5. Results suggest no significant structural changes in the receptor but do suggest that the four residues play different roles in defining ligand affinity and both intra- and intermolecular interactions play a role in defining ligand affinity. Thus, these results established two primary but necessary goals: (1) the baseline knowledge of perturbation of conserved interfacial residues for future reference and (2) the ability of the computational approach to identify the distal residues connecting to the interfacial residues. The data presented here provide the foundation for future experiments to identify the effects of distal residues that affect the specificity and affinity of BMP recognition. Protein-protein interactions are integral reactions in essentially all biological activities such as gene regulation and age-related development. Often, diseases are consequences of the alteration of these intermacromolecular interactions, which are thus recognized as a legitimate target for developing modulators for regulating biological functions. One approach is to design ligands that bind to the protein-protein interface. Another is to identify an allosteric site, an advantage of which is bypassing the potential challenge in competing for high-affinity interfacial interactions or a specific interface in a superassembly of multiple macromolecules. However, a challenge of this approach is identifying a site that is situated away from but functionally connected to the protein-protein interface.

IGF-1 Receptor Insufficiency Leads to Age-Dependent Attenuation of Osteoblast Differentiation.

In the current study, we determined the effects of IGF-1 receptor haploinsufficiency on osteoblast differentiation and bone formation throughout the lifespan. Bone mineral density was significantly decreased in femurs of male and female Igf1r(+/-) mice compared with wild-type mice. mRNA expression of osteoblast differentiation markers was significantly decreased in femurs and calvariae from Igf1r(+/-) mice compared with cells from wild-type mice. Bone morphogenetic protein-7-induced ectopic bone in Igf1r(+/-) mice was significantly smaller with fewer osteoblasts but more lipid droplets and had reduced expression of osteoblast differentiation markers compared with wild-type mice. In bone marrow cells from middle-aged and old wild-type and Igf1r(+/-) male mice, palmitate inhibited osteoblast markers expression. In cells from young wild-type male mice, palmitate did not inhibit marker expression, but in cells from young male Igf1r(+/-) mice, palmitate inhibited bone sialoprotein and osterix but not osteocalcin or type I collagen (TIC). In female wild-type mice, palmitate inhibited osteoblast markers expression in cells from young, middle-aged, and old mice except TIC in cells from middle-aged mice. Palmitate inhibited bone sialoprotein expression in cells from middle-aged and old female Igf1r(+/-) mice and osteocalcin, osterix, and TIC expression in young and middle-aged female Igf1r(+/-) mice but stimulated expression in cells from old female Igf1r(+/-) mice. We conclude that IGF-1 receptor haploinsufficiency results in a prolipid accrual phenotype in bone in association with inhibition of growth factor-induced osteoblast differentiation, a situation which may phenocopy age-related decreases in bone formation.

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

Protein kinase D1 is essential for bone acquisition during pubertal growth.

Bone formation and maintenance represents the summation of the balance of local and endocrine hormonal stimuli within a complex organ. Protein kinase D (PKD) is a member of the Ca(2+)/calmodulin-dependent kinase superfamily of serine/threonine kinases and has been described as the crossroads for the bone morphogenetic protein (BMP)-IGF-I signaling axis, which plays a major role in bone formation. The current study exploits the PKD1-deficient mouse model to examine the role of PKD in vivo in the skeleton. Dual-energy x-ray absorptiometry scan analysis of male and female pubescent mice demonstrated significantly decreased bone mineral density in the whole body and femoral bone compartments of PKD1 (+/-) mice, compared with their wild-type littermates. The body weight, nasal-anal length, and percentage body fat of the mice were not significantly different from their wild-type littermates. Cultured bone marrow stromal cells from PKD1 (+/-) mice demonstrated lower alkaline phosphatase activity in early differentiating osteoblasts and decreased mineralized nodule formation in mature osteoblasts. Quantitative RT-PCR analysis of osteoblast differentiation markers and osteoclast markers exhibited lower levels of expression in PKD1 (+/-) male mice than wild type. In female mice, however, only markers of osteoblast differentiation were reduced. PKD1 (+/-) mice also demonstrated a profound reduction in mRNA expression levels of BMP type II receptor and IGF-I receptor and in BMP-7 responsiveness in vitro. Together these data suggest that in mice, PKD1 action contributes to the regulation of osteoblastogenesis by altering gene expression with gender-specific effects on osteoclastogenesis, subsequently affecting skeletal matrix acquisition during puberty.

Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation.

A host-guest relationship in bone morphogenetic protein receptor-II defines specificity in ligand-receptor recognition.

One of the most intriguing questions confronting the bone morphogenetic protein family is the mechanism of ligand recognition, because there are more ligands than receptors. Crystal structures of two type II receptors, ActR-II and BMPR-II, are essentially identical, and a loop structure (A-loop) has been suggested to play a role in determining ligand specificity. A solution biophysical study showed mutations of several A-loop residues in these two receptors exert different ligand binding effects. Thus, the issues of mechanism of ligand recognition and specificity remain unresolved. We examined effects of mutations of residues Y40, G47, and S107 in BMPR-II. These residues are not identified as being in contact with the ligand in the BMP-7-BMPR-II complex but are found mutated in genetic diseases. They are likely to be useful in identifying their roles in differentiating the various BMP ligands. Spectroscopic probing revealed little mutation-induced structural change in BMPR-II. Ligand binding studies revealed that Y40 plays a significant role in differentiating three distinct ligands; G47 and S107 affect ligand binding to a lesser extent. The role of the A-loop in ActR-II or BMPR-II is dependent on the host sequence of the receptor extracellular domain (ECD) in which it is embedded, suggesting a host-guest relationship between the A-loop and the rest of the ECD. Computational analysis demonstrated a long-range connectivity between Y40, G47, and S107 and other locations in BMPR-II. An integration of these results on functional energetics and protein structures clearly demonstrates, for the first time, an intradomain communication network within BMPR-II.

Protein kinase D mediates the synergistic effects of BMP-7 and IGF-I on osteoblastic cell differentiation.

We previously showed that exogenous insulin-like growth factor-I (IGF-I) and bone morphogenetic protein-7 (BMP-7) synergistically stimulated osteoblast differentiation in fetal rat calvaria (FRC) cells. We have now shown that BMP-7 alone and the BMP-7 and IGF-I combination synergistically stimulated protein kinase D (PKD) phosphorylation at Ser744/748 and Ser916. Transfection of FRC cells with a constitutively active PKD stimulated marker expression, while transfection with a catalytically inactive PKD did not. Moreover, Gö6976, which inhibits protein kinase C (PKC) α and β1, blocked PKD phosphorylation and the synergistic action of the BMP-7 and IGF-I combination on osteoblast differentiation, whereas Gö6983, which inhibits PKCα, β, γ, δ, and ζ, did not. Our results suggest that the FRC cell differentiation induced by BMP-7 and the BMP-7 and IGF-I combination requires stimulation of PKD activity. Our results are consistent with a novel mechanism in which combined BMP-7 and IGF-I signaling activates upstream novel PKC(s), which then phosphorylates and activates PKD, leading to enhanced osteoblast differentiation.

Effects of cartilage-derived morphogenetic protein-3 on the expression of chondrogenic and osteoblastic markers in the pluripotent mesenchymal C3H10T1/2 cell line.

CDMP-3/GDF-7/BMP-12 treatment of pluripotent mesenchymal C3H10T1/2 cells resulted in a dose- and time-dependent change in cell morphology and in the expression of alkaline phosphatase, mRNA expression of osteocalcin, and bone sialoprotein, as well as mineralized bone nodule formation. CDMP-3 also stimulated Alcian Blue staining indicative of extracellular matrix formation without affecting aggrecan expression. CDMP-3 downregulated mRNA expression of BMP-4 and BMP-8A. CDMP-3 stimulated mRNA expression of ALK-1, ALK-2(ActR-IA), ALK-3(BMPR-IA), and ALK-4 without affecting that of ALK-6(BMPR-IB), ALK-7, and BMPR-II. These findings suggest that, under the experimental conditions studied, CDMP-3 induces the pluripotent mesenchymal C3H10T1/2 cells to express both chondrocytic and osteoblastic markers. The results further reveal potential complex interplay between the different bone morphogenetic proteins and their receptors in these processes.

In vitro and in vivo studies on the effects of bone morphogenetic protein-7 on human kidney and lung tumor cells.

Breast, kidney, lung, and prostate cancers are among the human cancers that show high propensity to form bone metastasis. Bone morphogenetic protein (BMP) -2 and -7 are two members of the BMP superfamily which show the most potent biological activity in stimulating bone differentiation and repair. These proteins have been used in clinical treatment of orthopedic diseases and have also been studied in different types of cancer. We report here detection of mRNA coding for three type I and one type II BMP receptors in G-402 kidney tumor cells and A-549 lung tumor cells, suggesting that these cells are responsive to BMPs. We then observed that BMP-7 inhibited cell proliferation of both cell lines in a protein concentration dependent manner in vitro. Additionally, when BMP-7-treated cells were implanted into the flank region of male nude mice, smaller tumors, compared to those formed with the untreated cells, were observed. Histological analysis showed that the masses formed at the site of implantation displayed significantly less number of tumors than the control and exhibited significant ectopic bone formation. These findings raise the possibility of BMP-7 as a therapeutic agent for kidney and lung cancers.

A comparative study on BMP-induced osteoclastogenesis and osteoblastogenesis in primary cultures of adult rat bone marrow cells.

Bone remodeling is a process consisting of bone formation and resorption. The present study compared the relative osteoclastic and osteoblastic potency of bone morphogenetic proteins (BMP)-2, -4, -5, -6, and -7 in primary murine bone marrow cultures. All five BMPs stimulated, to varying degree, formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in a time- and protein concentration-dependent manner. The TRAP staining intensity correlated positively with the number of nuclei per TRAP-positive cell and the mRNA levels of colony-stimulating factor (CSF-1), receptor activator of nuclear factor kappaB ligand (RANKL), TRAP, and cathepsin K. Under osteogenic conditions, all five BMPs stimulated AP activity and mineralized bone nodule formation in a protein concentration-dependent manner in the same primary murine bone marrow cell culture system. These findings should be useful in designing treatment strategies for bone regeneration.

Bone morphogenetic protein-7 regulates differentially the mRNA expression of bone morphogenetic proteins and their receptors in rat achilles and patellar tendon cell cultures.

Previous animal studies have suggested that certain bone morphogenetic proteins (BMPs) may be useful therapeutically in treating tendon healing. To better understand the relationship among the different BMPs in the healing process, we initiated the present study to examine the effects of a member of the BMP family, BMP-7 (also called Osteogenic Protein-1) on the temporal and spatial expression patterns of other BMPs and the BMP receptors in cell cultures of adult rat Achilles and Patellar tendons. Cultures from both tendon types expressed detectable but variable levels of biochemical markers characteristics of tendons. RNAs coding for type II collagen and transcription factors Six1, Scleraxis, and Tendin were detected in both types of cultures. Distinct patterns of expression of several BMP members and their receptors were observed in these cultured cells and BMP-7 exerted differential effects on their expression. The findings may have implications in the treatment of different tendon injuries with BMPs.

Characterization of ligand-binding properties of the human BMP type II receptor extracellular domain.

ActR-IIA, ActR-IIB, and BMPR-II are low-affinity type II receptors that bind bone morphogenetic proteins (BMPs) in the same overall manner. The binding of BMPs by ActR-IIs has been analyzed structurally and functionally, but no detailed analysis of BMPR-II has been reported. The objective of this study was to determine ligand-binding epitopes and specificity determinants in two regions, the hydrophobic patch and the A-loop of the BMPR-II extracellular domain (ECD). A series of alanine-substituted variants was generated using a recently published X-ray structure of the unliganded form of the ovine BMPR-II ECD as a guide. These variants were characterized using one-dimensional NMR and functional activity assays with BMP-2, BMP-7 and GDF-5 as ligands. The results showed that alanine substitutions of conserved residues W85 and Y113 within the hydrophobic patch of the ECD differentially perturbed BMP ligand binding without disrupting receptor folding, suggesting that they are critical determinants for ligand binding and ligand specificity. Our results further revealed that the nonconserved residue L69 in the hydrophobic patch contributes to ligand-binding activity and specificity. Mutations of several residues within the A-loop resulted in minimal effects on the binding of the different BMP ligands. Overall, these observations identify several amino acid residues that play different roles in BMPR-II and ActR-II and thereby enable BMPR-II and ActR-IIs to bind different subclasses of BMP ligands.

A fluorescence study of type I and type II receptors of bone morphogenetic proteins with bis-ANS (4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid).

Crystallography studies on several members of the bone morphogenetic protein (BMP) receptors suggested that hydrophobic regions in these proteins play an important role in their structure and function. In the present study, the environment sensitive fluorescent probe 4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid (bis-ANS) was used to study the hydrophobic regions of the extracellular domain of the type I and II receptors for bone morphogenetic proteins (ecBMPR-IB and ecBMPR-II). A single bis-ANS binding site per receptor molecule was found for both receptors, but the two receptors interacted with bis-ANS with distinctive characteristics. A significant shift in the emission maximum from 498 to 510 nm was detected when bis-ANS binds ecBMPR-IB, but a negligible change in the emission maximum was observed when the dye binds ecBMPR-II. Under identical reaction conditions, the maximum fluorescence intensities of the probe (I(max)) for the ecBMPR-IB and -II are 4.0 and 6.2 x 10(4) arbitrary units, respectively. The probe binds to ecBMPR-IB and -II with K(d)=11.0 and 17.5 microM, respectively. The bis-ANS modified site on both receptor types was not readily accessible to acrylamide quenching. Fluorescence energy transfer experiments further revealed close proximity between the tyrosine (in ecBMPR-IB) and the tryptophan residue (in ecBMPR-II) and the respective bis-ANS binding site in these receptors. The binding of bis-ANS did not alter the ligand binding activity of ecBMPR-IB, but enhanced that of ecBMPR-II. These results show that the bis-ANS-modified hydrophobic site on the ecBMPR-IB and -II molecules plays a different functional role.

Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

C-type natriuretic peptide enhances osteogenic protein-1-induced osteoblastic cell differentiation via Smad5 phosphorylation.

In the present study, we examined the hypothesis that the C-type natriuretic peptide (CNP) enhances osteogenic protein-1 (OP-1) action in stimulating osteoblastic cell differentiation in primary cultures of fetal rat calvaria cell (FRC). CNP enhanced synergistically the OP-1-induced Alkaline Phosphatase (AP) activity and mineralized bone nodule formation in a dose- and time-dependent manner. To examine possible mechanism of the synergy between OP-1 and CNP, the expression levels of key BMP receptors and signaling molecules were examined. Western blot analysis showed that BMPR-IB and -II receptor protein expression was not affected by CNP alone, but was stimulated by OP-1 alone. The combination of OP-1 and CNP did not further increase their protein levels. The Runx2 protein expression level was not altered by CNP alone, but was elevated by OP-1 alone, and was slightly reduced by the combination. The Smad5 protein expression level was slightly decreased by CNP alone, but was stimulated by OP-1 alone, and was not further stimulated by the combination. Smad5 phosphorylation was not stimulated by CNP alone, but was stimulated significantly by OP-1 alone. The combination of OP-1 and CNP further stimulated the OP-1-induced Smad5 phosphorylation. Thus, one mechanism of the observed synergy between OP-1 and CNP involves enhancement of the Smad5 phosphorylation.

Co-transfection with the osteogenic protein (OP)-1 gene and the insulin-like growth factor (IGF)-I gene enhanced osteoblastic cell differentiation.

Previous studies from this laboratory showed that the action of Osteogenic Protein-1 (OP-1, BMP-7) on osteoblastic cell differentiation could be enhanced by other protein factors, such as Insulin-like Growth Factor (IGF)-I. In the present study, we examined the effects of co-transfection with a combination of the OP-1 and the IGF-I gene on osteoblastic cell differentiation. The results first showed that fetal rat calvaria (FRC) cells transfected with the OP-1 gene under the control of the cytomegalovirus (CMV) promoter showed substantial production of the OP-1 protein. Transfected FRC cells also showed a DNA concentration-dependent increase in alkaline phosphatase (AP) activity, an osteoblastic cell differentiation marker. Von Kossa-positive nodules, a hallmark of bone formation in long-term cultures of bone-derived cells, were also observed in the transfected cells after 26 days in culture, whereas none were observed in control cells. Co-transfection of FRC cells with the combination of the OP-1 and the IGF-I gene resulted in a synergistic stimulation of AP activity. The increase was DNA dose-dependent. The current data show that transfection of OP-1 gene into osteoblastic cells stimulates osteoblastic cell differentiation in vitro. The study further demonstrates the feasibility of employing gene transfer of a second gene in combination with an OP-1 vector to synergistically enhance OP-1 activity.

Cartilage-derived morphogenetic proteins induce osteogenic gene expression in the C2C12 mesenchymal cell line.

Cartilage-derived morphogenetic protein-1, -2, and -3 (CDMP-1, -2, and -3) are members of the bone morphogenetic protein (BMP) family and have been shown to exhibit a variety of biological activities. In the present study, effects of these CDMPs on the temporal and spatial expression of genes in the pluripotent mesenchymal cell line C2C12 were examined. Cells cultured in the presence of CDMPs lost the characteristic elongated shape of myoblasts. At the molecular level, CDMP treatment did not change the mRNA expression of MyoD, aggrecan, Six1, and tendin. Scleraxis mRNA level was reduced by CDMP treatment. CDMP-1 and -3, but not CDMP-2, stimulated expression of osteogenic markers, such as alkaline phosphatase (AP), osteocalcin (OC), BSP, and type I collagen, in a dose- and time-dependent manner. With few exceptions, the three CDMPs changed, with different potencies, the expression profile of different members of the BMP family in a similar temporal pattern. Except at the late phase of treatment, CDMP treatment did not change the expression of ActR-IA, BMPR-IA, BMPR-IB, BMPR-II, and ALK-7 mRNAs. Based on the current data, the CDMPs appear to be able to stimulate the C2C12 cells to differentiate into the osteoblast pathway.

Cartilage-derived morphogenetic proteins enhance the osteogenic protein-1-induced osteoblastic cell differentiation of C2C12 cells.

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) induces differentiation of the pluripotent mesenchymal cell line C2C12 into osteoblastic cells. OP-1 also alters the steady-state levels of messenger RNA (mRNA) encoding for the cartilage-derived morphogenetic proteins (CDMPs) in C2C12 cells. In the present study, the effects of exogenous CDMPs on bone cell differentiation induced by OP-1 in C2C12 cells were examined. Exogenous CDMP-1, -2, and -3 synergistically and dose-dependently enhanced OP-1 action in stimulating alkaline phosphatase (AP) activity and osteocalcin (OC) mRNA expression. AP staining studies revealed that the combination of OP-1 and CDMP enhanced OP-1 action by stimulating those cells that had responded to OP-1 and not by activating additional cells. The combination did not change the mRNA expression of the BMPs and their receptors. CDMP-1 enhanced the suppression of the OP-1-induced expression of the myogeneic differentiation regulator MyoD. CDMP-1 and OP-1 alone stimulated Smad5 protein expression, but the combination of OP-1 and CDMP-1 stimulated synergistically Smad5 protein expression. Thus, one mechanism of the observed synergy involved enhancement of the induced Smad5 protein expression. At the same protein concentration, CDMP-1 is most potent in enhancing OP-1 activity in inducing osteoblastic cell differentiation of C2C12 cells. CDMP-3 is about 80% as potent as CDMP-1, and CDMP-2 is the least potent (about 50% of CDMP-1).

Identification of an osteogenic protein-1 responsive element in the aggrecan promoter.

Previous studies have demonstrated that osteogenic protein-1 (OP-1), a member of the bone morphogenetic protein (BMP) subfamily of the TGF-beta superfamily, stimulates aggrecan synthesis. To investigate transcriptional regulation of the aggrecan gene by OP-1, we constructed a clone containing a 1 kb region of the 5'-upstream sequence of the mouse aggrecan gene fused to the promoter-less luciferase reporter gene in pGL2-Basic vector. A series of promoter deletion constructs were also generated. Studies of the promoter activity of these DNA constructs in transient-transfected cells revealed that deletion of a 33 bp region rendered the promoter unresponsive to OP-1, BMP-6, and CDMP-1 without affecting BMP-2 and TGF-beta1 responsiveness. Thus, the expression of the mouse aggrecan gene in response to BMPs appears to be the result of a unique combination of different cis-acting elements.