Samuel Varghese

Transforming growth factor β1 inhibits collagenase 3 expression by transcriptional and post-transcriptional mechanisms in osteoblast cultures

Transforming growth factor (TGF) β1 is an autocrine regulator of bone cell function. We demonstrated that TGF β1 enhances bone collagen synthesis, but its effects on collagen degradation are not well characterized. We tested the effects of TGF β1 on rat collagenase 3 expression in cultures of osteoblast‐enriched cells from fetal rat calvariae (Ob cells). Treatment with TGF β1 at 0.4 nM decreased steady state collagenase mRNA levels after 2 to 24 h. This dose‐dependent effect was observed at TGF β1 concentrations of 4 pM to 1.2 nM, and was accompanied by decreased levels of immunoreactive procollagenase. The protein synthesis inhibitor cycloheximide increased collagenase transcripts, but did not prevent the effect of TGF β1 on collagenase mRNA levels. TGF β1 accelerated the decay of collagenase mRNA in transcriptionally arrested Ob cells. In addition, TGF β1 decreased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. TGF β1 enhanced the expression of tissue inhibitors of metalloproteinases (TIMP) 1 and 3 and caused a modest decrease of TIMP 2 mRNA levels. In conclusion, TGF β1 decreases interstitial collagenase transcripts and protease levels in Ob cells by transcriptional and post‐transcriptional mechanisms, and this effect may contribute to its actions on bone matrix. J. Cell. Physiol. 170:145–152, 1997.

Effects of serum from children with newly diagnosed Crohn disease on primary cultures of rat osteoblasts

Objectives We propose that Crohn disease (CD) decreases bone formation via circulating inflammatory mediators. We therefore examined the effects of serum from newly diagnosed, untreated children with CD on osteoblasts in culture and the role of interleukin-6 (IL-6), a cytokine present in excess in active CD that also has direct effects on bone. Methods Bone mineral density was measured by dual x-ray absorptiometry. Primary cultures of rat osteoblasts were treated with serum from patients with CD and healthy controls. We measured expression of osteoblast proliferation, viability, differentiation markers, and mineralized nodule formation. Neutralizing antibodies were used to inhibit the effects of IL-6 present in serum. Results We studied 24 children with CD (14 male) and 31 controls (15 male). Spine bone mineral density was lower in patients with CD (Z score, −0.8 ± 0.9 vs. 0.0 ± 1.0 for controls;P < 0.05). Nodule formation was markedly decreased in osteoblasts treated with CD serum. However, CD serum did not affect osteoblast proliferation or viability. Expression of proteins characteristic of mature osteoblasts—osteocalcin and alkaline phosphatase—was reduced. Unlike our results in a model of intact bone, neutralization of IL-6 did not inhibit the effects of CD serum. Addition of IL-6 to control serum to match serum concentrations in CD had no effect either. Conclusions CD serum affects osteoblast function and probably differentiation in vitro, suggesting a mechanism by which CD may affect bone formation. IL-6 by itself is not sufficient to cause these effects and probably needs a cofactor present in intact bone.

Leukemia inhibitory factor and oncostatin M stimulate collagenase-3 expression in osteoblasts

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) have multiple effects on skeletal remodeling. Although these cytokines modestly regulate collagen synthesis in osteoblasts, their effects on collagenase expression and collagen degradation are not known. We tested whether LIF and OSM regulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from fetal rat calvariae. LIF and OSM increased collagenase-3 (MMP-13) mRNA and immunoreactive protein levels in a time- and dose-dependent manner. LIF and OSM enhanced the rate of transcription of the collagenase gene and stabilized collagenase mRNA in transcriptionally arrested cells. LIF and OSM failed to regulate the expression of gelatinase A (MMP-2) and B (MMP-9). LIF and OSM modestly stimulated the expression of TIMP-1 but did not alter the expression of TIMP-2 and -3. In conclusion, LIF and OSM stimulate collagenase-3 and TIMP-1 expression in osteoblasts, and these effects may be involved in mediating the bone remodeling actions of these cytokines.Leukemia inhibitory factor (LIF) and oncostatin M (OSM) have multiple effects on skeletal remodeling. Although these cytokines modestly regulate collagen synthesis in osteoblasts, their effects on collagenase expression and collagen degradation are not known. We tested whether LIF and OSM regulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from fetal rat calvariae. LIF and OSM increased collagenase-3 (MMP-13) mRNA and immunoreactive protein levels in a time- and dose-dependent manner. LIF and OSM enhanced the rate of transcription of the collagenase gene and stabilized collagenase mRNA in transcriptionally arrested cells. LIF and OSM failed to regulate the expression of gelatinase A (MMP-2) and B (MMP-9). LIF and OSM modestly stimulated the expression of TIMP-1 but did not alter the expression of TIMP-2 and -3. In conclusion, LIF and OSM stimulate collagenase-3 and TIMP-1 expression in osteoblasts, and these effects may be involved in mediating the bone remodeling actions of these cytokines.

Alendronate Stimulates Collagenase 3 Expression in Osteoblasts by Posttranscriptional Mechanisms

Bisphosphonates inhibit bone resorption by reducing osteoclastic cell number and activity. Alendronate is a nitrogen‐containing bisphosphonate analog used in the treatment of postmenopausal osteoporosis. The effects of alendronate in osteoclasts are well documented; however, there is limited information on the actions of alendronate in osteoblasts (Obs). In this study, we investigated the effects of alendronate at concentrations of 1‐100 μM on the synthesis of collagenase 3 or matrix metalloproteinase 13 (MMP‐13) and tissue inhibitors of MMPs (TIMPs) 1, 2, and 3 in primary Ob‐enriched cells from 22‐day‐old fetal rat calvariae. Alendronate at concentrations higher than 10 μM markedly stimulated the synthesis of collagenase messenger RNA (mRNA) and immunoreactive protein in Obs. Alendronate did not stimulate the transcriptional rate of the collagenase 3 gene. However, in transcriptionally arrested cells, alendronate prolonged the half‐life of collagenase transcripts. Alendronate did not alter the expression of TIMP 1 and 2, but modestly stimulated the expression of TIMP 3. The actions of alendronate in Obs suggest potential additional effects in bone remodeling.

Bone morphogenetic protein-2 suppresses collagenase-3 promoter activity in osteoblasts through a runt domain factor 2 binding site.

Transforming growth factor‐β (TGFβ) superfamily of growth factors, which include bone morphogenetic proteins (BMPs), have multiple effects in osteoblasts. In this study, we examined the regulation of collagenase‐3 promoter activity by BMP‐2 in osteoblast‐enriched (Ob) cells from fetal rat calvariae. BMP‐2 suppressed the activity of a −2 kb collagenase‐3 promoter/luciferase recombinant in a time‐ and dose‐dependent manner. The BMP‐2 effect on the collagenase‐3 promoter was further tested in several collagenase‐3 promoter deletion constructs and it was narrowed down to a −148 to −94 nucleotide segment of the promoter containing a runt domain factor 2 (Runx2) site at nucleotide −132 to −126. The effect of BMP‐2 was obliterated in a collagenase‐3 promoter/luciferase construct containing a mutated Runx2 (mRunx2) sequence indicating that the Runx2 site mediates the BMP‐2 response. Electrophoretic mobility shift assays, using nuclear extracts from control and BMP‐2‐treated Ob cells, indicated that the Runx2 protein is a component of the specific DNA–protein complex formed on the Runx2 site and that the BMP‐2 effect may be associated with minor protein modifications rather than major changes in the composition of specific proteins interacting with the Runx2 site. We confirmed that other members of the TGFβ family can down‐regulate the collagenase‐3 promoter by showing that TGFβ1 also suppresses the promoter activity in a time‐ and dose‐dependent manner. In conclusion, this study demonstrates that BMP‐2 and TGFβ1 suppress collagenase‐3 promoter activity in osteoblasts and establishes a link between BMP‐2 action and collagenase‐3 expression via Runx2, a major regulator of osteoblast formation and function.

Transcriptional regulation of collagenase-3 by interleukin-1 alpha in osteoblasts

Interleukin‐1 (IL‐1)α is an autocrine/paracrine agent of the skeletal tissue and it regulates bone remodeling. Collagenase‐3 or matrix metalloproteinase (MMP)‐13 is expressed in osteoblasts and its expression is modulated by several cytokines including IL‐1α. Because the molecular mechanism of increased synthesis of collagenase‐3 in bone cells by IL‐1α is not known, we investigated if collagenase‐3 expression by IL‐1α in osteoblasts is mediated by transcriptional or post‐transcriptional mechanisms. Exposure of rat osteoblastic cultures (Ob cells) to IL‐1α at concentrations higher than 0.5 nM increased the synthesis of collagenase‐3 mRNA up to eightfold and the secretion of immunoreactive protein up to 21‐fold. The effects of IL‐1α on collagenase‐3 were time‐ and dose‐dependent. Although prostaglandins stimulate collagenase‐3 expression, stimulation of collagenase‐3 in Ob cells by IL‐1α was not mediated through increased biosynthesis of prostaglandins. The half‐life of collagenase‐3 mRNA from control and IL‐1α‐treated Ob cells was similar suggesting that the stabilization of collagenase‐3 mRNA did not contribute to the increase in collagenase‐3. However, IL‐1α stimulated the rate of transcription of the collagenase‐3 gene by twofold to fourfold indicating regulation of collagenase‐3 expression in Ob cells at the transcriptional level. Stimulation of collagenase‐3 by IL‐1α in osteoblasts may in part mediate the effects of IL‐1α in bone metabolism.