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

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

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

Using S -adenosyl- L -[Me- 14 C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of added Escherichia coli tRNA and in its presence. [Me- 14 C] tRNA was purified from both systems and its [Me- 14 C] base composition determined. The endogenous formation of [Me- 14 C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereas E. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me- 14 C] 1-methyl guanine and [Me- 14 C] N 2 -methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me- 14 C] N 2 2 -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me- 14 C] N 2 -methyl and N 2 2 -dimethyl guanine and significantly higher proportions of [Me- 14 C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45399/1/11064_2004_Article_BF00966229.pd