The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use of cultures of primary keratinocytes from wild-type and PPAR␤/␦ −/− mice, that such signals including TNF-␣ and IFN-␥, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requires up-regulation of the PPAR␤/␦ gene via the stress-associated kinase cascade, which targets an AP-1 site in the PPAR␤/␦ promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenous PPAR␤/␦ ligands, which are essential for PPAR␤/␦ activation and action. Activated PPAR␤/␦ regulates the expression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes to cell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin of PPAR␤/␦ +/+ and PPAR␤/␦ +/− mice.[ Peroxisome proliferator-activated receptors (PPARs), which control many cellular and metabolic processes, are members of the superfamily of ligand-inducible transcription factors known as nuclear receptors. Three isotypes called PPAR␣ (NR1C1), PPAR␤/␦ (NR1C2), and PPAR␥ (NR1C3) (Nuclear Receptors Nomenclature Committee 1999) have been identified in vertebrates. They display differential tissue distribution, suggesting that each of them fulfills specific functions. PPAR␣ and PPAR␥ play important roles in lipid homeostasis and in inflammation (Desvergne and Wahli 1999). In contrast, the exact functions of PPAR␤ remain an enigma. Although fatty acids can bind and activate PPAR␤, studies on this isotype have so far been impeded by the lack of information about the nature of its physiological ligands and by its remarkably broad tissue distribution. Recently however, PPAR␤ was implicated in reverse cholesterol transport (Oliver et al. 2001), in oligodendrocyte maturation and in membrane sheet formation (Saluja et al. 2001). In addition, PPARs may play an important role in skin development, as PPAR␣ ligands can accelerate fetal rat epidermal development (Hanley et al. 1998). In epidermis, the three PPAR isotypes are expressed during development, but their levels decrease to become undetectable in the interfollicular keratinocytes 5-9 d after birth (Michalik et al. 2001). However, the expression of both PPAR␣ and PPAR␤ is reactivated upon proliferative stimuli, such as treatment with the phorbol ester TPA or hair plucking and at the wound edges after skin injury. Although PPAR␤ −/− and PPAR␤ +/− mice are not affected by apparent skin defects, they display an increased hyperplasic response to TPA treatment (Peters et al. 2000;Michalik et al. 2001). More interestingly, wound closure is delayed in PPAR␤ +/− mice as compared with wild-type animals (Michalik et al. 2001). Taken together, these observations suggest that PPAR␤ may play an important role in skin, particularly in stress situations.The epidermis in which keratinocyte is the predominant cell type is characterized by a lifelong polarized pattern of epithelial growth and cell differ...

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.

In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.