Geranylgeranyl Pyrophosphate Promotes Profibrotic Factors and Collagen-Specific Chaperone HSP47 in Fibroblasts

Abstract

Fibrosis, characterised by excessive extracellular matrix deposition, contributes to both organ failure and significant mortality worldwide. Whereas fibroblasts are activated into myofibroblasts, marked by phenotypic factors such as α-smooth muscle actin (α-SMA), periostin, fibroblast activation protein (FAP) and heat shock protein 47 (HSP47), the cellular processes of trans-differentiation for fibrosis development remain poorly understood. Herein, we hypothesised that the molecular signalling of geranylgeranyl pyrophosphate (GGPP), a crucial biochemical molecule for protein prenylation, is essential in the regulation of profibrotic mechanisms for fibroblast-to-myofibroblast activation. To test this hypothesis, we demonstrated pharmacological inhibition of geranylgeranyl pyrophosphate synthase (GGPS1) significantly decreased TGF-β1-dependent myofibroblast differentiation assessed by reduced α-SMA, periostin, FAP and HSP47 expression. Exogenous GGPP in the presence of GGPS1 inhibition restored TGF-β1-induced differentiation, supporting posttranslational requirements of GGPP modification during myofibroblast differentiation. Selective inhibition of either geranylgeranyl transferase or farnesyl transferase significantly impacted TGF-β1-induced myofibroblast α-SMA and HSP47 expression. The importance of protein prenylation as a key regulator of myofibroblast differentiation was remarkably revealed by an unexpected decrease in HSP47 expression. In contrast, direct HSP47 inhibition not only suppressed TGF-β1-induced α-SMA expression but surprisingly could not be rescued using exogenous GGPP. A selective role for the ER-resident chaperone HSP47 expression downstream of GGPP was suggested when the effects of GGPS1 inhibition on periostin expression were counteracted by GGPP and geranylgeranyl transferase inhibition. Taken together, our findings underscore for the first time the functional role of cholesterol synthesis-independent GGPP-dependent pathway in fibroblast-to-myofibroblast transition and open new potential therapeutic targets for antifibrosis therapies.

Keywords: HSP47; differentiation; fibroblasts; geranylgeranyl pyrophosphate.

FIGURE 1

GGPS1 inhibition decreases TGF‐β1‐induced fibroblast–myofibroblast trans differentiation. (A) Immunoblot showing incubation of TGF‐β1 (5 ng/mL) for 72 h increased the expression of α‐SMA without any significant difference in the GAPDH expression while inhibition of GGPS1 by co‐administration of DGBP (10 μM) significantly decreased the TGF‐β1‐induced α‐SMA expression. Lower panel displays the bar graph representing the mean values of corresponding band intensities of the above blot. (B) Immunoblot showing incubation of TGF‐β1 (5 ng/mL) either in the presence or absence of DGBP (10 μM) for 72 h did not affect the expression of either geranylgeranyl pyrophosphate synthase 1 (GGPS1) enzyme or β‐tubulin. Lower panel displays the bar graph representing the mean values of corresponding band intensities of the above blot. (C) Immunoblot showing expression of FAP, HSP47 and GAPDH either in the presence of TGF‐β1‐ or co‐administration of DGBP. Right side bar graph depicts the respective mean values of band intensities. DGBP has significantly (p < 0.05) decreased the TGF‐β1‐induced increase in the expression of FAP. Similarly, the expression of HSP47 was also significantly (p < 0.01) decreased by DGBP in the TGF‐β1 group. One way ANOVA, N = 3; *p < 0.05 and **p < 0.01 considered significant.

FIGURE 2

Cell morphology and viability under various treatment conditions. Representative bright field images (10×) of fibroblasts/myofibroblasts showing viability of the cells at various conditions following treatments for 72 h with TGF‐β1 (5 ng/mL), inhibition of GGPS1 by DGBP (10 μM), co‐administration of GGPP (25 μM), inhibition of geranylgeranyl transferase by GGTI‐298 (10 μM), inhibition of farnesyl transferase by FTI‐277 (5 μM), inhibition of Rho GTPase by rhosin (10 μM), inhibition of Rho‐associated kinase (ROCK) by Y27632 (10 μM), inhibition of G protein βγ subunit‐dependent signalling by gallein (10 μM), direct inhibition of HSP47 by HY124817 (10 μM) or co‐administration with GGP (25 μM). D0: Day 0; D3: Day 3, following plating of cells.

FIGURE 3

Cholesterol synthesis‐independent pathway mechanisms involved in fibroblast–myofibroblast trans‐differentiation. Top panel shows the representative immunoblot of all treatment conditions and bottom panel depicts the bar graph with respective mean values of band intensities. Exogenous administration of GGPP (25 μM) prevented the DGBP (10 μM)—induced inhibition of α‐SMA, a marker of activated myofibroblasts. In the presence of GGPP (TGF + DGBP + GGPP), there was no significant difference in the α‐SMA expression compared to TGF group. Inhibition of GGT by GGTI‐298 (5 μg/mL) significantly decreased the TGF‐induced α‐SMA expression, and inhibition of farnesyl transferase by FTI‐277 (5 μM) did not significantly affect the TGF‐β1‐induced α‐SMA expression. Similarly, either inhibition of Rho GTPase by rhosin (10 μM) or inhibition of G protein βγ subunit‐dependent signalling by gallein (10 μM) did not significantly affect the TGF‐β1‐induced increased α‐SMA expression. However, inhibition of Rho‐associated kinase (ROCK) by Y27632 (10 μM) significantly decreased the TGF‐β1‐induced α‐SMA expression; and direct inhibition of HSP47 by HY124817 (10 μM) significantly inhibited the TGF‐β1‐induced α‐SMA expression that was not sensitive to exogenous administration of GGPP (25 μM) of mevalonate (300 μM). One‐way ANOVA, N = 3; *p < 0.05 or **p < 0.01 considered significant.

FIGURE 4

Signalling mechanisms involved in the TGF‐β1‐induced upregulation of HSP47. Top panel shows the representative immunoblot of all treatment conditions and bottom panel depicts the bar graph with respective mean values of band intensities. Exogenous administration of GGPP (25 μM) prevented the DGBP (10 μM)—induced inhibition of HSP47. In the presence of GGPP (TGF + DGBP + GGPP), there was no significant difference in the HSP47 expression compared to TGF group. Inhibition of GGT by GGTI‐298 (5 μg/mL) significantly decreased the TGF‐β1‐induced HSP47 expression‐like inhibition of farnesyl transferase by FTI‐277 (5 μM). However, inhibition of Rho GTPase by rhosin (10 μM), inhibition of Rho‐associated kinase (ROCK) by Y27632 (10 μM) or inhibition of G protein βγ subunit‐dependent signalling by Gallein (10 μM) did not significantly affect the TGF‐β1‐induced increased HSP47 expression. Direct inhibition of HSP47 by HY124817 (10 μM) significantly inhibited the TGF‐β1‐induced HSP47 expression, which was not sensitive to exogenous administration of GGPP (25 μM) of mevalonate (300 μM). One way ANOVA, N = 6; *p < 0.05, **p < 0.01 and ***p < 0.001 considered significant.

FIGURE 5

Signalling mechanisms regulating TGF‐β1‐induced periostin expression. Top panel shows the representative immunoblot of all treatment conditions and bottom panel depicts the bar graph with respective mean values of band intensities. Exogenous administration of GGPP (25 μM) prevented the DGBP (10 μM)—induced inhibition of periostin. In the presence of GGPP (TGF + DGBP + GGPP), there was no significant difference in the periostin expression compared to TGF group. Inhibition of GGT by GGTI‐298 (5 μg/mL) significantly decreased the TGF‐β1‐induced periostin expression unlike inhibition of farnesyl transferase by FTI‐277 (5 μM). However, inhibition of Rho GTPase by rhosin (10 μM), inhibition of Rho‐associated kinase (ROCK) by Y27632 (10 μM) or inhibition of G protein βγ subunit‐dependent signalling by gallein (10 μM) did not significantly affect the TGF‐β1‐induced increased periostin expression. Direct inhibition of HSP47 by HY124817 (10 μM) did not significantly affect the TGF‐β1‐induced periostin expression, which was not sensitive to exogenous administration of GGPP (25 μM) of mevalonate (300 μM). One way ANOVA, N = 6; *p < 0.05 and **p < 0.01 considered significant.

FIGURE 6

Schema: GGPP signalling in fibroblast/myofibroblast differentiation. In fibroblasts, 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase located in the ER converts HMG‐CoA into mevalonate that is converted by series of enzymatic steps into farnesyl pyrophosphate (FPP). Geranylgeranyl pyrophosphate is synthesised by GGPS1 (geranylgeranyl disphosphate synthase (1) from FPP. Geranylgeranyl transferase (GGTase 1) catalyses the prenylation of various proteins. Through subsequent potential signalling steps, profibrotic process including collagen processing and α‐SMA (α‐smooth muscle actin) expression happens with the involvement of HSP47, while periostin expression occurs independent of HSP47. Solid arrows: Direct step; Broken arrows: Multiple steps involved.