Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes

Background During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes. Results Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition. Conclusion Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate

The Month Of June Is A Song Of Love

https://digitalcommons.library.umaine.edu/mmb-vp/2134/thumbnail.jp

A Little Bit Of Irish

https://digitalcommons.library.umaine.edu/mmb-vp/4650/thumbnail.jp

Say Boys! I\u27ve Found A Girl

https://digitalcommons.library.umaine.edu/mmb-vp/6401/thumbnail.jp

Impaired Bone Formation in Transgenic Mice Resulting from Altered Integrin Function in Osteoblasts

AbstractTo determine the role of integrins in mature osteoblasts in vivo, we expressed in transgenic mice a dominant-negative integrin subunit (β1-DN) consisting of the β1 subunit cytoplasmic and transmembrane domains, driven by the osteoblast-specific osteocalcin promoter. Immature osteoblasts isolated from transgenic animals differentiated normally in vitro until the osteocalcin promoter became active; thereafter they detached from the substratum, suggesting that β1-DN was impairing adhesion in mature osteoblasts. Transgenic animals had reduced bone mass, with increased cortical porosity in long bones and thinner flat bones in the skull. At 35 days, the rate of bone formation was reduced in cortical bone, and the parietal bones were 45% thinner than in wild-type animals. Active osteoblasts were less polar and had larger areas of cytoplasm with intracellular stores of matrix molecules. Osteocyte lacunae appeared normal around the cell body but did not have normal canilicular structures. At 90 days, the parietal bone of transgenic males was of normal width, suggesting that the original defect in matrix deposition had been repaired or compensated for. In contrast, transgenic females still had decreased bone mass in the parietal bone at 90 days. The decreased bone mass in TG females was accompanied by increased staining for osteoclast activity, suggesting that there was a sex-specific defect in mature animals

Solution mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSC) on PLGA by promote the formation of a calcium phosphate rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, we tested whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSC, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell derived factors which promoted distant MSC differentiation. Thus PLGABG composites demonstrates significant potential as a bone replacement material

Of How That Woman Could Cook / music by Grace Le Boy; words by Gus Kahn

Cover: a drawing of a woman cooking and baking; photo inset of Sam Chip and Mary Marble; Publisher: Jerome H. Remick and Co. (New York)https://egrove.olemiss.edu/sharris_c/1076/thumbnail.jp

Disparate Osteogenic Response of Mandible and Iliac Crest Bone Marrow Stromal Cells to Pamidronate

OBJECTIVE Long-term administration of intravenous bisphosphonates like pamidronate is associated with jaw osteonecrosis but axial and appendicular bones are unaffected. Pathogenesis of bisphosphonate-associated jaw osteonecrosis may relate to skeletal-site specific effects of bisphosphonates on osteogenic differentiation of bone marrow stromal cells (BMSCs) of orofacial and axial/appendicular bones. This study evaluated and compared skeletal site-specific osteogenic response of mandible (orofacial bone) and iliac crest (axial bone) human BMSCs to pamidronate. MATERIALS AND METHODS Mandible and iliac crest BMSCs from six normal healthy volunteers were established in culture and tested with pamidronate to evaluate and compare cell survival, osteogenic marker alkaline phosphatase, osteoclast differentiation in co-cultures with CD34+ hematopoietic stem cells, gene expression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin, and in vivo bone regeneration. BRESULTS Mandible BMSCs were more susceptible to pamidronate than iliac crest BMSCs based on decreased cell survival, lower alkaline phosphatase production and structurally less organized in vivo bone regeneration. Pamidronate promoted higher RANKL gene expression and osteoclast recruitment by mandible BMSCs. CONCLUSION Mandible and iliac crest BMSC survival and osteogenic differentiation are disparately affected by pamidronate to favor dysregulated mandible bone homeostasis

Smad-Runx interactions during chondrocyte maturation

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. Clinical Relevance: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy