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Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis., (2005 Wiley-Liss, Inc.)

We have investigated the mechanisms by which all-trans retinoic acid (ATRA) causes growth inhibition of ovarian carcinoma cells. As a model, we have studied the CAOV3 cell line, which is sensitive to ATRA, and the SKOV3 cell line, which is resistant. We have found that treatment of CAOV3 cells with ATRA causes a 5-10 fold increase in the protein level of the cyclin dependent kinase inhibitor p27/Kip1. p27/Kip1 protein upregulation is important in ovarian carcinoma as primary tumors are frequently found lacking this protein. The increase in p27/Kip1 is detected by day 3 of ATRA treatment of CAOV3 cells, and is maximal by day 5. Messenger RNA levels of p27/Kip1 do not change in CAOV3 cells following ATRA treatment, however, we have shown that p27/Kip1 mRNA is more stable in ATRA treated CAOV3 cells. Conversely, the ATRA resistant cell line SKOV3 fails to show p27/Kip1 accumulation. Interestingly, the SCF component protein SKP2 appears to be decreased in CAOV3 cells treated with ATRA. We have also shown that the ATRA dependent increase in p27/kip1 protein in CAOV3 cells leads to a decrease in the kinase activity of cyclin dependent kinase 4 (CDK4) following ATRA treatment. Finally, we found that CAOV3 cells stably transfected with a p27/kip1antisense construct, which express lower levels of p27/kip1 following ATRA treatment, and have a higher CDK4 kinase activity are less sensitive to ATRA induced growth suppression. Taken together our data suggest ATRA-induced growth inhibition in CAOV3 ovarian carcinoma cells involves modulation of the CDK inhibitor p27/kip1.

Aortic vascular smooth muscle cells (VSMC) were used to study the effect of age on responses to high glucose concentrations or the cytokine, tumor necrosis factor-alpha (TNF-alpha). Activator protein-1 (AP-1) binding to DNA increased more in VSMC from old versus young rats (P < 0.02) and was related to increased expression of its components, c-Fos, Fra-1, and JunD. The relationship to upstream signals, i.e., activities of mitogen-activated protein kinases (MAPK), was studied using antibodies to total and phosphorylated forms of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. High glucose and TNF-alpha increased ERK phosphorylation more in old (P < 0.05); whereas only TNF-alpha induced JNK activation in young (P < 0.04). PD98059, a MEK inhibitor, attenuated AP-1 activation, lowered c-Fos and Fra-1 protein levels and reduced cell number and cells positive for proliferating cell nuclear antigen in old. We concluded that age differentially influenced activation of signaling pathways in VSMC exposed to high glucose or TNF-alpha. This may contribute to the increased risk for vascular disease associated with aging and diabetes mellitus (DM).