Crosstalk between Shh and TGF-β Signaling in Cyclosporine-Enhanced Cell Proliferation in Human Gingival Fibroblasts

BACKGROUND: Immunosuppressant cyclosporine-A induces gingival hyperplasia, which is characterized by increased fibroblast proliferation and overproduction of extracellular matrix components and regulated by transforming growth factor-beta (TGF-β). The TGF-β and Sonic hedgehog (Shh) pathways both mediate cell proliferation. Crosstalk between these pathways in cancer has recently been proposed, but the hierarchical pattern of this crosstalk remains unclear. In normal fibroblasts, a TGF-β-stimulating Shh pattern was observed in induced fibrosis. However, Shh pathway involvement in cyclosporine-enhanced gingival proliferation and the existence of crosstalk with the TGF-β pathway remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Cyclosporine enhanced mRNA and protein levels of TGF-β and Shh in human gingival fibroblasts (RT-PCR and western blotting). A TGF-β pathway inhibitor mitigated cyclosporine-enhanced cell proliferation and an Shh pathway inhibitor attenuated cyclosporine-enhanced proliferation in fibroblasts (MTS assay and/or RT-PCR of PCNA). Exogenous TGF-β increased Shh expression; however, exogenous Shh did not alter TGF-β expression. The TGF-β pathway inhibitor mitigated cyclosporine-upregulated Shh expression, but the Shh pathway inhibitor did not alter cyclosporine-upregulated TGF-β expression. CONCLUSIONS/SIGNIFICANCE: The TGF-β and Shh pathways mediate cyclosporine-enhanced gingival fibroblast proliferation. Exogenous TGF-β increased Shh expression, and inhibition of TGF-β signaling abrogated the cyclosporine-induced upregulation of Shh expression; however, TGF-β expression appeared unchanged by enhanced or inhibited Shh signaling. This is the first study demonstrating the role of Shh in cyclosporine-enhanced gingival cell proliferation; moreover, it defines a hierarchical crosstalk pattern in which TGF-β regulates Shh in gingival fibroblasts. Understanding the regulation of cyclosporine-related Shh and TGF-β signaling and crosstalk in gingival overgrowth will clarify the mechanism of cyclosporine-induced gingival enlargement and help develop targeted therapeutics for blocking these pathways, which can be applied in pre-clinical and clinical settings.