His occurs remains largely unknown. Drug-induced gingival overgrowth is usually a side effect of three classes of medications: phenytoin is an anti-seizure drug, nifedipine is really a calcium channel blocker, and cyclosporine A is definitely an immunosuppressant. Our laboratory has located that CCN2/CTGF is highly expressed in phenytoin induced gingival overgrowth, whereas it’s not expressed in cyclosporine A induced overgrowth [Hong et al., 1999; Uzel et al., 2001]. CCN2/CTGF is discovered at intermediate levels in nifedipine induced gingival overgrowth [Uzel et al., 2001]. As phenytoin induced lesions would be the most fibrotic, and cyclosporine induced lesions are usually not Kainate Receptor Antagonist Formulation fibrotic but hugely inflamed, we reasoned that CCN2/CTGF most likely contributes to fibrosis in phenytoin induced lesions. At the same time, we have identified no impact of CCN2/CTGF on collagen mRNA levels in gingival fibroblast cultures, whereas CCN2/CTGF properly increased collagen deposition in these cultures [Hong et al., 1999]. The major goal of the present study, therefore, was to investigate structure/function relationships of CCN2/CTGF inside the stimulation of collagen deposition. Also, we investigated the role of numerous integrins in mediating effects of CCN2/CTGF on collagen deposition. So that you can achieve these goals we CBP/p300 Inhibitor review developed a relatively speedy assay for collagen deposition in gingival fibroblasts. These findings supply new insights in to the mechanisms by which CCN2/CTGF contributes to fibrosis in gingival tissues, and may possibly furthermore ultimately deliver new therapeutic methods to address fibrotic disease in other tissues at the same time.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCell CultureMATERIALS AND METHODSHuman recombinant CTGF/CCN2 was kindly provided by FibroGen Corporation, South San Francisco, and was created in a baculovirus expression system. The N-terminal half of CTGF/ CCN2 (containing module 1 two) plus the C-terminal half (containing module 3 four) and affinity purified goat polyclonal antibodies recognizing these portions of CTGF/CCN2 have been also generously provided. The N-terminal and C-terminal halves of CTGF have been affinity purified following partial digestion of full-length CTGF with chymotrypsin, which specifically cleaves the molecule amongst module 2 and module 3. The polyclonal antibody against fulllength recombinant human CTGF was purified by affinity chromatography. N-terminal or Cterminal specific polyclonal antibodies were ready from the affinity purified polyclonal antibody by purification on affinity columns made from C-terminal or N-terminal halves, respectively. Specificity with the purified polyclonal antibodies for the N-terminal or C-terminal half fragments had been confirmed by Western blotting. Human recombinant TGF-1 was bought from Peprotech, Rocky Hill, NJ. Sirius Red powder was obtained from Chroma, M ster, Germany. Anti- integrin monoclonal neutralizing antibodies have been bought from Chemicon, Temecula, CA: anti-1 (catalogue MAB2253Z, clone B44), anti-3 (catalogue # MAB2023Z, cloneB3A), and M (catalogue # MAB1380, clone ICRF44), plus the anti-6 integrin neutralizing antibody clone GoH3 (catalogue # 0796) was purchased from Immunotech, Coulter, France. The anti-integrin IIb antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA (catalog # sc19963). If antibody formulations contained azide, these samples had been completely dialyzed against cold PBS before use. All other reagents were bought from Sigma Invitrogen.
