Tissue-specific Mechanisms for CCN2/CTGF Persistence in Fibrotic Gingiva

Abstract

Prostaglandin E 2 blocks transforming growth factor TGF ␤1-induced CCN2/CTGF expression in lung and kidney fibroblasts. PGE 2 levels are high in gingival tissues yet CCN2/CTGF expression is elevated in fibrotic gingival overgrowth. Gingival fibroblast expression of CCN2/CTGF in the presence of PGE 2 led us to compare the regulation of CCN2/CTGF expression in fibroblasts cultured from different tissues. Data demonstrate that the TGF␤1-induced expression of CCN2/CTGF in human lung and renal mesangial cells is inhibited by 10 nM PGE 2 , whereas human gingival fibroblasts are resistant. Ten nM PGE 2 increases cAMP accumulation in lung but not gingival fibroblasts, which require 1 M PGE 2 to elevate cAMP. Micromolar PGE 2 only slightly reduces the TGF␤1-stimulated CCN2/CTGF levels in gingival cells. EP2 prostaglandin receptor activation with butaprost blocks the TGF␤1-stimulated expression of CCN2/CTGF expression in lung, but not gingival, fibroblasts. In lung fibroblasts, inhibition of the TGF␤1-stimulated CCN2/CTGF by PGE 2 , butaprost, or forskolin is due to p38, ERK, and JNK MAP kinase inhibition that is cAMP-dependent. Inhibition of any two MAPKs completely blocks CCN2/ CTGF expression stimulated by TGF␤1. These data mimic the inhibitory effects of 10 nM PGE 2 and forskolin that were dependent on PKA activity. In gingival fibroblasts, the sole MAPK mediating the TGF␤1-stimulated CCN2/CTGF expression is JNK. Whereas forskolin reduces TGF␤1-stimulated expression of CCN2/CTGF by 35% and JNK activation in gingival fibroblasts, micromolar PGE 2 -stimulated JNK in gingival fibroblasts and opposes the inhibitory effects of cAMP on CCN2/CTGF expression. Stimulation of the EP3 receptor with sulprostone results in a robust increase in JNK activation in these cells. Taken together, data identify two mechanisms by which TGF␤1-stimulated CCN2/CTGF levels in human gingival fibroblasts resist down-regulation by PGE 2 : (i) cAMP cross-talk with MAPK pathways is limited in gingival fibroblasts; (ii) PGE 2 activation of the EP3 prostanoid receptor stimulates the activation of JNK.

Transforming Growth Factor-β1 (TGFβ1) Stimulates Connective Tissue Growth Factor (CCN2/CTGF) Expression in Human Gingival Fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent Mechanism

“…TGF␤1 and LPA Stimulate Similar Levels of JNK1 Phosphorylation in Human Gingival Fibroblasts-We previously demonstrated that JNK1 phosphorylation induced by TGF␤1 is essential for maximal CCN2/CTGF expression (40). We hypothesize that, like TGF␤1, LPA might stimulate JNK1 activation.…”

“…Activation of the c-Jun N-terminal kinase 1 (JNK1) is essential for maximal expression of CCN2/CTGF in response to TGF␤1 in gingival fibroblasts (40). We hypothesized that Rho family GTPases were required in mediating the TGF␤1-induced activation of JNK.…”

“…We hypothesized that the combination of partial JNK inhibition with forskolin (40), and the independent inhibitory effects of lovastatin directed at Rho family GTPases, could together more fully reduce CCN2/CTGF protein levels in response to TGF␤1 in gingival fibroblasts. To test this, gingival cell cultures were treated with 10 M forskolin alone or in combination with 20 M lovastatin for 12 h in serum-free media.…”

“…We have previously used the Chariot reagent and have determined that it is effective at introducing proteins directly into primary gingival fibroblasts (40). Gingival fibroblasts were then incubated for 12 h with 5 g of C3 protein/ Chariot reagent.…”

“…Primary human gingival fibroblasts are unique in their resistance to the inhibition of the TGF␤1-induced expression of CCN2/CTGF by prostaglandin E 2 (PGE 2 ), whereas in primary human renal mesangial cells and IMR90 human lung fibroblasts PGE 2 potently blocks CCN2/CTGF expression stimulated by TGF␤1 (40). Although it has been demonstrated that renal fibroblasts require activated RhoA to promote CCN2/CTGF expression in response to LPA and TGF␤ (8,9), the role of the Rho family GTPases in mediating the TGF␤1-stimulated expression of CCN2/CTGF has yet to be determined in gingival cells.…”