Protease nexin-1 messenger RNA levels are not affected by serum or interferon beta in cultured systemic sclerosis fibroblasts.

The aim of the present study was to analyze the effect of serum and human recombinant beta interferon (rIFNbeta) treatment on PN-1 mRNA levels in cultured dermal fibroblasts obtained from the skin of healthy donors and from lesional skin of systemic sclerosis (SSc) patients with the limited (CREST syndrome) or the diffuse form of SSc. Total RNA was isolated from fibroblasts derived from the skin of healthy individuals and from lesional skin of patients with CREST syndrome and the diffuse form of SSc cultured under different conditions (1% or 10% serum-supplemented medium) and treated with 500 IU/ml of rIFNbeta. PN-1 gene expression was assessed by Northern blot analysis. We detected variable PN-1 mRNA levels in normal control fibroblasts as well as in SSc fibroblasts under the different culture conditions (1% or 10% serum-supplemented medium). Accumulated PN-1 mRNA levels found in normal cultured fibroblasts were similar to or even higher than in SSc fibroblasts. PN-1 messenger levels were not significantly altered by IFNbeta treatment in normal or SSc cultured fibroblasts despite the presence of an IFN-stimulated responsive element (ISRE) in the promoter of the PN-1 gene. Our findings suggest that PN-1 expression in SSc fibroblasts at the mRNA level requires further investigation in a large number of SSc patients to better characterize the role of this serpin in the pathogenesis of SSc. We conclude that the transcriptional regulation of PN-1 is not associated with IFNbeta, an antifibrotic cytokine naturally produced by fibroblasts.

A nuclear target for interleukin-1alpha: interaction with the growth suppressor necdin modulates proliferation and collagen expression.

There is growing evidence for the intracellular role of cytokines and growth factors, but the pathways by which these activities occur remain largely obscure. Previous work from our laboratory identified the constitutive, aberrant expression of the 31-kDa IL-1 alpha precursor (pre-IL-1 alpha) in the nuclei of fibroblasts from the lesional skin of patients with systemic sclerosis (SSc). We established that pre-IL-1 alpha expression was associated with increased fibroblast proliferation and collagen production. Further investigation has led to the identification of a mechanism by which nuclear expression of pre-IL-1 alpha affects fibroblast growth and matrix production. By using a yeast two-hybrid method, we found that pre-IL-1 alpha binds necdin, a nuclear protein with growth suppressor activity. We mapped the region of pre-IL-1 alpha responsible for necdin binding and found it to be localized near the N terminus, a region that is present on pre-IL-1 alpha, but not the mature 17-kDa cytokine. Expression studies demonstrated that pre-IL-1 alpha associates with necdin in the nuclei of mammalian cell lines and regulates cell growth and collagen expression. Our results provide the first evidence, to our knowledge, of a nuclear target for pre-IL-1 alpha. Based on these findings, we propose that the constitutively up-regulated expression of pre-IL-1 alpha in the nuclei of SSc fibroblasts up-regulates proliferation and matrix production of SSc fibroblasts through binding necdin, and by counteracting its effects on cell growth and collagen production.