Figure 6. Role of PKC in the induction by TSH of IL-6.

(A) Confluent orbital fibroblasts and fibrocytes were treated with TSH alone or in combination with GFX (10 µM) for 16 h. Media were subjected to an IL-6 ELISA. Data are expressed as mean ± SD of triplicate independent determinations. (##, P<0.01 vs untreated controls **, P<0.01 vs TSH alone). In 3 separate experiments, GFX inhibited TSH-provoked IL-6 expression by 64.2±5.1% and 65.9±4.8% in fibroblasts and fibrocytes, respectively. (B) Cultures were treated with nothing, bTSH, 8-Br-cAMP (1 mM), PMA (50 ng/mL) or the combination indicated for 16 h. Media were analyzed for IL-6 content (**, P<0.01 vs untreated controls). (C). Cellular protein from three strains of each cell type were subjected to Western blot analysis of PKCµ and PKCβII. (D) RNA was extracted from the cell types indicated and subjected toRT-PCR for PKCµ and PKCβII mRNA by the C_T method. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. (E) Cultures were treated with nothing, bTSH (5 mIU/mL), or PMA (50 ng/mL) for 30 min, harvested, and proteins analyzed by Western blot for PKCµ, pPKCµ (Ser 916), PKCβII, and pPKCβII (Ser 660). Results are from a single experiment, representative of three performed. (F) Targeting siRNAs and their scrambled counterparts were transfected into sub- confluent monolayers as described in Experimental Procedures. After 48 h, they were treated with nothing or bTSH (5 mIU/mL) for 16 h. Media were collected and subjected to an IL-6 ELISA. Data are expressed as the mean ± SD of three independent determinations from a single experiment, representative of three performed. **, p<0.01 vs TSH-treated cultures transfected with control siRNA. In 3 separate experiments, PKCµ siRNA inhibited TSH-provoked IL-6 by 48.5±1% in fibroblasts and PKCβII siRNA inhibited TSH-induced IL-6 by 59± % in fibrocytes.