Biomedical subjects

Baruch Frenkel

Publications and source records attributed to Baruch Frenkel.

12 recordsLinked to original sources

PubMed2006

Peripheral cannabinoid receptor, CB2, regulates bone mass.

The endogenous cannabinoids bind to and activate two G protein-coupled receptors, the predominantly central cannabinoid receptor type 1 (CB1) and peripheral cannabinoid receptor type 2 (CB2). Whereas CB1 mediates the cannabinoid psychotropic, analgesic, and orectic effects, CB2 has been implicated recently in the regulation of liver fibrosis and atherosclerosis. Here we show that CB2-deficient mice have a markedly accelerated age-related trabecular bone loss and cortical expansion, although cortical thickness remains unaltered. These changes are reminiscent of human osteoporosis and may result from differential regulation of trabecular and cortical bone remodeling. The CB2(-/-) phenotype is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors. CB2 is expressed in osteoblasts, osteocytes, and osteoclasts. A CB2-specific agonist that does not have any psychotropic effects enhances endocortical osteoblast number and activity and restrains trabecular osteoclastogenesis, apparently by inhibiting proliferation of osteoclast precursors and receptor activator of NF-kappaB ligand expression in bone marrow-derived osteoblasts/stromal cells. The same agonist attenuates ovariectomy-induced bone loss and markedly stimulates cortical thickness through the respective suppression of osteoclast number and stimulation of endocortical bone formation. These results demonstrate that the endocannabinoid system is essential for the maintenance of normal bone mass by osteoblastic and osteoclastic CB2 signaling. Hence, CB2 offers a molecular target for the diagnosis and treatment of osteoporosis, the most prevalent degenerative disease in developed countries.

Animals↗

PubMed2005

ERK1/2-activated de novo Mapkapk2 synthesis is essential for osteogenic growth peptide mitogenic signaling in osteoblastic cells.

In osteoblasts, the mitogen-activated protein kinases ERK1/2 and p38 as well as the cAMP-response element-binding protein (CREB) have been implicated in the regulation of proliferation and differentiation. The osteogenic growth peptide (OGP) is a 14-mer bone cell mitogen that increases bone formation and trabecular bone density and stimulates fracture healing. OGP-(10-14) is the physiologically active form of OGP. Using gene array analysis, real-time reverse transcription-PCR, and immunoblot and DNA synthesis assays we show here that in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures the OGP-(10-14) mitogenic signaling is critically dependent on de novo synthesis of mitogen-activated protein kinase-activated protein kinase 2 (Mapkapk2) mRNA and protein. The increase in Mapkapk2 occurs following short term (5-60 min) stimulation of ERK1/2 activity by OGP-(10-14); phosphorylation of p38 remains unaffected. Downstream of Mapkapk2, CREB is phosphorylated on Ser(133) leading to its enhanced transcriptional activity. That these events are critical for the OGP-(10-14) mitogenic signaling is demonstrated by blocking the effects of OGP-(10-14) on the ERK1/2 pathway, Mapkapk2, CREB, and DNA synthesis using the MEK inhibitor PD098059. The OGP-(10-14) stimulation of CREB transcriptional activity and DNA synthesis is also blocked by Mapkapk2 siRNA. These data define a novel mitogenic signaling pathway in osteoblasts whereby ERK1/2 stimulation of CREB phosphorylation and transcriptional activity as well as DNA synthesis are critically dependent on de novo Mapkapk2 synthesis.

Animals↗

PubMed2005

Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells.

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.

Adenocarcinoma↗

PubMed2005

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo.

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, -3 and +4, are usually such that support initiation (A-3 = 42%, G-3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)-3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R-3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R-3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R-3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R-3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.

Animals↗

PubMed2005

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20-binding enhancer.

OBJECTIVE: Glucocorticoids are widely used for the management of rheumatoid arthritis. Osteoporosis is a major side effect of glucocorticoid therapy and is attributable to inhibition of bone formation. We developed an osteoblast culture system in which glucocorticoids strongly inhibit development of the osteoblast phenotype, including expression of the bone-specific osteocalcin (OC) gene. Using this gene as a model, the goal of this study was to discover glucocorticoid-sensitive transcriptional mechanisms in osteoblasts. METHODS: Dexamethasone (DEX; 1 microM) was administered to murine MC3T3-E1 osteoblastic cultures under conditions that inhibit mineralized extracellular matrix formation and OC messenger RNA levels by >10-fold. Because standard (short-term) transient transfection assays with OC promoter-reporter constructs did not recapitulate the strong DEX-mediated repression, mapping of OC negative glucocorticoid response elements (GREs) was performed initially by stable transfection and then with long-term transient transfection assays. Transcription factor binding to the OC negative GRE was studied by electrophoretic mobility shift assays. RESULTS: Several-fold repression of OC-luciferase constructs was recapitulated in stable and long-term transient transfection assays, in which the transfected cells were allowed to progress to a sufficiently advanced developmental stage. Analysis of a 5' promoter deletion series mapped an OC negative GRE to a 15-bp G/C-rich motif (-161/-147) located just upstream of the binding site for the osteoblast master transcription factor Runx2. Oligonucleotides encompassing this element and MC3T3-E1 cell extracts formed a protein-DNA complex that contained an Egr/Krox family member(s). Complex formation was competed by either an oligonucleotide containing 2 consensus Egr motifs or by anti-Egr2/Krox20 antibodies. Three copies of this Krox-binding element conferred 20-fold transcriptional activation on the 147-bp basal OC promoter in osteoblasts, and the enhancer activity was inhibited by DEX. Enhancer activity was not observed in 10T1/2 fibroblasts unless these cells were cotransfected with Runx2. CONCLUSION: An Egr2/Krox20-binding site located immediately upstream of the Runx2 site of the mouse OC promoter was identified as an enhancer in osteoblasts, whose activity is repressed by glucocorticoids. Sequence similarity suggests that such a mechanism is likely operative in both murine and human cells. Because glucocorticoids inhibit Egr2/Krox20 expression in osteoblasts, and because trabecular bone formation is arrested in Egr2/Krox20-knockout mice, the inhibition of Egr2/Krox20 activity likely contributes to glucocorticoid-induced osteoporosis.

Animals↗

PubMed2004

Glucocorticoids inhibit the transcriptional activity of LEF/TCF in differentiating osteoblasts in a glycogen synthase kinase-3beta-dependent and -independent manner.

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation-related cell cycle is associated with a decrease in beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser(473) within minutes, and this inhibition was also observed after 12 h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and Wnt signaling pathways. Consistent with a role for Wnt signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt signaling and the osteoblast differentiation-related cell cycle.

3T3 Cells↗

PubMed2004

ChIP Display: novel method for identification of genomic targets of transcription factors.

Novel protein-DNA interactions in mammalian cells are traditionally discovered in the course of promoter studies. The genomic era presents opportunities for the reverse; namely, the discovery of novel target genes for transcription factors of interest. Chromatin immunoprecipitation (ChIP) is typically used to test whether a protein binds to a candidate promoter in living cells. We developed a new method, ChIP Display (CD), which allows genome-wide unbiased identification of target genes occupied by transcription factors of interest. Initial CD experiments pursuing target genes for RUNX2, an osteoblast master transcription factor, have already resulted in the identification of four genes that had never been reported as targets of RUNX2. One of them, Osbpl8, was subjected to mRNA and promoter-reporter analyses, which provided functional proof for its regulation by RUNX2. CD will help to assemble the puzzle of interactions between transcription factors and the genome.

Animals↗

PubMed2004

Gene expression profiling of glucocorticoid-inhibited osteoblasts.

Glucocorticoid (GC) treatment for the management of autoimmune and inflammatory diseases is associated with decreased bone formation and increased risk for fracture. In MC3T3-E1 cell cultures, 0.1-1 microM dexamethasone (DEX) arrests development of the osteoblast phenotype when administration commences at a commitment stage around the time of confluency. To gain new insights into GC-induced osteoporosis, we performed microarray-based gene expression analysis of GC-arrested MC3T3-E1 cultures, 2.5 days after the administration of DEX. Of the >12 000 transcripts interrogated, 74 were up-regulated and 17 were down-regulated by at least 2.5-fold (P < or = 0.05). Some of these genes, such as Mmp13, Serum/GC-regulated kinase and Tieg, have previously been reported as GC-responsive. Others are shown here for the first time to respond to GCs. DEX strongly repressed Krox20/Egr2 at both the mRNA and the protein level. This is especially significant because mice lacking this transcription factor develop osteoporosis. The data also suggest that the bone morphogenetic protein (BMP) pathway, which is involved in regulating bone mass, and other pathways that influence BMP signaling, are abrogated by GCs: (i) DEX increased the mRNA levels of the BMP antagonists Follistatin and Dan; (ii) DEX increased the levels of p21 Rasgap3 and Ptpn16/MKP-1 mRNAs, negative regulators of the MAP kinase pathway; and (iii) DEX decreased Cox mRNA levels. DEX also increased thrombospondin mRNA levels, which negatively regulate bone mass in vivo, as well as the adipocytic marker Fkbp51. These and other observations disclose novel gene targets, whose regulation by GCs in osteoblasts may shed light on and provide new therapeutic approaches to osteoporosis.

3T3 Cells↗

PubMed2003

Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without alteration of Runx2.

Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 microm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.

Alkaline Phosphatase↗

PubMed2003

Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures.

UNLABELLED: The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 microM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids. INTRODUCTION: Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts. MATERIALS AND METHODS: MC3T3-E1 cells were treated with dexamethasone (DEX; 1 microM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). RESULTS: DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX. CONCLUSIONS: Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of CCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.

Animals↗

PubMed2003

Role of apoptosis in glucocorticoid-induced osteoporosis and osteonecrosis.

Bone disease is a side effect of concern regarding chronic glucocorticoid (GC) administration. Most GC-treated patients exhibit a process of bone loss, frequently leading to osteoporosis, with increased fracture risk, especially in spinal vertebrae. Some GC-treated patients will develop osteonecrosis, a disease with distinct clinical and histopathological features, most often occurring underneath the articular surface of the femoral head. Remarkably, both of these GC-induced bone diseases are associated with osteoblast and osteocyte apoptosis, which is emerging as a potential primary pathogenic mechanism. Here, we review the evidence for osteoblast and osteocyte apoptosis in GC-induced bone disease and highlight current debates: (1) With recent reports describing the antiapoptotic effect of GCs in some in vitro osteoblast models, and with the known adverse effects of GCs on osteoblast cell cycle and differentiation, could the in vivo osteoblast apoptosis be an indirect rather than a direct effect of GCs? (2) What is the pathogenic relationship between GC-induced osteoporosis and osteonecrosis? Could the latter be a mere manifestation of the former? and (3) How does apoptosis fit into the traditional concept of ischemia as a key etiology in osteonecrosis? Regardless of the answers, recent studies with cells, animals, and humans point out bone cell apoptosis as a potential target in the design of treatment for GC-induced bone disease.

Animals↗

PubMed2002

Glucocorticoids inhibit cell cycle progression in differentiating osteoblasts via glycogen synthase kinase-3beta.

Differentiating osteoblasts in culture undergo a commitment stage, during which cobblestone-like cells grow to high density past confluency. In contrast to earlier proliferative stages, the cell cycle during this commitment stage is inhibited by glucocorticoids (GC). Chronic GC treatment also impedes mineral deposition if steroid administration commences early enough during commitment. This study defines a role for glycogen synthase kinase-3beta (GSK3beta) and its target, c-Myc, in the GC-sensitive osteoblast persistent cell cycle. c-Myc levels decreased as cells reached confluence, but then increased during growth to high density. GC administration at this stage resulted in down-regulation of c-Myc. This was accompanied by GC-mediated attenuation of GSK3beta Ser(9) inhibitory phosphorylation and increased GSK3beta kinase activity. Down-regulation of c-Myc was attributable to enhanced Thr(58) phosphorylation, leading to accelerated degradation. In contrast, GC did not inhibit the c-Myc synthesis rate or the level of beta-catenin, a transcriptional coactivator of c-myc. The attenuated cell cycle and the reduced c-Myc level were returned to control levels by specific inhibition of GSK3beta using lithium chloride. These results suggest that tonal GSK3beta repression at the cobblestone stage of osteoblast differentiation permits osteoblast growth to high density. GC interfere with this growth-permissive axis by GSK3beta activation, resulting in c-Myc down-regulation and impediment of the G(1)/S cell cycle transition.

Animals↗