cell cycle ͉ proliferation ͉ Wnt ͉ corepressor T he process of skin carcinogenesis involves a series of transitional events, ranging from hyperplasia, dysplasia, and papilloma to invasive squamous cell carcinoma. The current model indicates that the progression of benign lesions to malignancy depends on the cell type targeted by these mutations (1, 2). Indeed, recent evidence has indicated that the bulge region of the hair follicles contains selfrenewing, slow-cycling stem cells, which give rise to transient amplifying cells and several differentiated cell lineages in the interfollicular epidermis (3, 4). The progression of skin carcinogenesis can be modified by mutations involving Tgf␤1, Smad3, ubiquitin ligase Fbxw7/Cdc4, or Pten (5-9), suggesting that a genetic network synergizes with activated Ras mutation to promote the initiation or malignant transformation of skin tumors (10). Another important regulator of skin carcinogenesis is cyclin D1. Overexpression of cyclin D1 increases the propensity of skin carcinogenesis, whereas loss of cyclin D1 reduces tumor formation (11,12). One mechanism to control cyclin D1 expression is through transcriptional regulation via the Wnt/␤-catenin signaling pathway (13,14), which consists of a core set of highly evolutionarily conserved proteins that have wide-ranging effects on gene expression affecting proliferation, migration, pluripotency, morphogenesis, and tumorigenesis (15-17). In the epidermis, targeted deletion of ␤-catenin during embryogenesis results in severe abnormalities in cell fate determination and maintenance of hair follicle formation (18). In contrast, mutations that activate ␤-catenin function lead to skin tumorigenesis in both humans and mice (19, 20).Homeodomain interacting protein kinase (HIPK) contains three distinct members that regulate apoptosis, cell growth, and proliferation. HIPK2 has been identified as a transcriptional corepressor for homeodomain proteins NK3 and Brn3a (21,22). Previous results have indicated that Brn3a and HIPK2 regulate a delicate balance of gene expression that controls programmed cell death in sensory and dopamine neurons (22)(23)(24). Both HIPK1 and HIPK2 can interact with human p53 and, under DNA damage conditions, activate p53 by promoting phosphorylation and acetylation (25)(26)(27). Contrary to the predicted function, HIPK1-deficient mice show a higher resistance to the development of DMBA-induced skin tumors (27). Recent evidence, however, has demonstrated a functional redundancy between HIPK1 and HIPK2 during development, which could compensate for the loss of HIPK1 (28). Indeed, HIPK2 has a wide range of functions, including promoting the degradation of CtBP in response to UV-induced DNA damage (29) and regulating cell proliferation through the Wnt signaling pathway (30). Furthermore, HIPK2 can interact with several proteins containing the high-mobility group I (HMGI), a domain highly conserved in transcription factors in the lymphoid enhancer-binding factor 1/T cell factor (LEF1-TCF) family (31).In this work, we ...

Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose-and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation. 7,8 and osteogenic differentiation of human mesenchymal stem cells. 9,10 Given that osteoprogenitors are the precursors to osteoblasts, the survival and differentiation of these cells is crucial to the process of bone formation in the implant bed during exposure to wear particles.Osteoprogenitor differentiation is regulated by the transcription factors Runx2, osterix, Dlx5, and Msx2, and the transcriptional activator b-catenin. 11 Runx2 dictates the expression of key osteoblast proteins such as alkaline phosphatase, collagen, and osteocalcin, and is expressed in bone cells throughout the osteogenic lineage. 11 Osterix lies downstream of Runx2 and is required for the differentiation of pre-osteoblasts into mature osteoblasts. 11,12 Dlx5 is a homeobox domain transcription factor that also regulates osteogenesis [13][14][15] and is involved in the response of osteogenic cells to BMP-2. [16][17][18] Msx2, another homeodomain transcription factor, is a functional antagonist and repressor of Dlx5-induced osteogenesis. 19-23 b-Catenin, a transcriptional activator of the canonical Wnt signaling pathway, associates with the DNA-binding proteins TCF and LEF, and stimulates Runx2 transcription. 11,24,25 Expression of these transcription factors is normally required for the proper differentiation of osteoprogenitor cells.Our group has previously shown that PMMA particles inhibit the osteogenic differentiation of murine MC3T3-E1 ...

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.