Depletion of protein kinase C? in normal and scleroderma lung fibroblasts has opposite effects on tenascin expression

Abstract

Objective To determine whether the extracellular matrix protein tenascin‐C (TN‐C) is overexpressed in lung fibroblasts from systemic sclerosis (SSc) patients, the molecular mechanisms regulating TN‐C secretion in SSc and normal lung fibroblasts, and how these processes might contribute to lung fibrosis in SSc patients. Methods TN‐C secretion by SSc and normal fibroblasts was compared in vivo (in bronchoalveolar lavage [BAL] fluid) and in vitro (in culture medium). The ability of thrombin to induce TN‐C was confirmed at both the protein and the messenger RNA (mRNA) level. The role of protein kinase Cε (PKCε) in the expression of TN‐C was evaluated by determining the effects of thrombin on PKCε levels and by directly manipulating PKCε levels via the use of antisense oligonucleotides. Results BAL fluid from SSc patients contained high levels of TN‐C, whereas that from normal subjects contained little or no TN‐C. In vitro, SSc lung fibroblasts expressed much higher amounts of TN‐C than did normal lung fibroblasts. Consistent with the idea that thrombin is a physiologic inducer of TN‐C, thrombin stimulated TN‐C mRNA and protein expression in both SSc and normal lung fibroblasts by a mechanism that required proteolytic cleavage of the thrombin receptor. Surprisingly, thrombin treatment and antisense oligonucleotide–mediated depletion of PKCε indicated that TN‐C expression is regulated via opposite signaling mechanisms in SSc and normal cells. In SSc lung fibroblasts, thrombin decreased PKCε levels, and the decreased PKCε induced TN‐C secretion; in normal fibroblasts, thrombin increased PKCε levels, and the increased PKCε induced TN‐C secretion. Normal and SSc lung fibroblasts also differed in the subcellular localization of PKCε, both before and after thrombin treatment. Conclusion These studies are the first to demonstrate that thrombin is a potent simulator of TN‐C in lung fibroblasts and that PKCε is a critical regulator of TN‐C protein levels in these cells. Furthermore, our results indicate that both the regulation of PKCε levels by thrombin and the regulation of TN‐C levels by PKCε are defective in SSc lung fibroblasts.

Opposing Effects of Protein Kinase Cα and Protein Kinase Cϵ on Collagen Expression by Human Lung Fibroblasts Are Mediated via MEK/ERK and Caveolin-1 Signaling

“…In summary, these observations further support the idea that ERK phosphorylation and collagen expression are tightly linked. In accord with our previous studies (11,12), these observations also suggest that the PKC⑀ signaling pathway functions aberrantly in SLF.…”

“…PKC Expression/Activity-To inhibit the expression of various isoforms of PKC, cells were treated with appropriate antisense oligodeoxynucleotides (ODN) and control sense ODN for 48 h as described previously (11) and then shifted to serum-free medium. The next morning the culture medium and cell layer were harvested.…”

“…In contrast, PKC⑀ functions abnormally in scleroderma lung fibroblasts (SLF). Depleting PKC⑀ in normal lung fibroblasts (NLF) inhibits expression of the extracellular matrix protein tenascin-C, whereas depleting PKC⑀ in SLF promotes tenascin-C expression (11). SLF are deficient in PKC⑀ and are sensitive to curcumin-induced apoptosis, whereas NLF are insensitive to curcumin (12).…”

Signaling pathways regulating intercellular adhesion molecule 1 expression by endothelin 1: Comparison with interleukin‐1β in normal and scleroderma dermal fibroblasts

“…The ratio of expression of these 2 isoforms differs substantially in scleroderma dermal fibroblasts, with greater PKC␦ expression (Figure 6D). Reduced expression of PKC in scleroderma lung fibroblasts has been reported previously (34,35). .…”

Inhibitory Effects of the Dual Endothelin Receptor Blocker Bosentan on Thrombin-Activated Lung Fibroblasts Isolated from Scleroderma Patients

“…Thrombin, besides its importance in thrombosis and hemostasis, also has several important functions in the stimulation of platelets, leukocytes, endothelial cells, smooth muscle cells, and lung fibroblasts [6,8,9]. The multiple effects of thrombin on cells include regulation of proliferation, stimulation and expression of various cytokines, and regulation of extracellular matrix (ECM) [9][10][11]. Previously, we demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the PAR-1 receptor and via a PKC-dependent pathway [6].…”