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The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

The eosinophilia-myalgia syndrome was associated with the ingestion of L-tryptophan products containing a number of contaminants, one of which has been identified as 1,1'-ethylidene-bis-(L-tryptophan) (EBT), also known as peak E or peak 97. In earlier studies, we demonstrated that EBT induces inflammation and fibrosis in dermal and subcutaneous tissue of C57BL/6 mice. Others have shown EBT to be a potent stimulus for fibroblast activation and collagen synthesis in vitro, and dermal tissue from EMS patients reveals evidence of enhanced collagen gene expression. In the present study using Northern blot analysis and in situ hybridization, we demonstrate enhanced expression of genes for types I, III, and VI collagen in the dermis and subcutis of C57BL/6 mice treated with EBT for 3-21 days. Increased type I procollagen mRNA was noted on day 6 of EBT treatment and was followed by enhanced expression of type III and VI procollagen mRNA at day 21. L-Tryptophan, free of contaminants associated with the eosinophilia-myalgia syndrome epidemic, increased dermal collagen mRNA to a lesser extent than did EBT. Increased procollagen gene expression was accompanied by evidence of enhanced TGF-beta 1 expression in the dermis and subcutis. This animal model provides additional evidence for EBT as a causal agent of the eosinophilia-myalgia syndrome and should prove useful in the study of the pathogenesis of that syndrome.