A potential role for protease nexin 1 overexpression in the pathogenesis of scleroderma
David Strehlow, … , Karen Strehlow, Joseph H. Korn
David Strehlow, … , Karen Strehlow, Joseph H. Korn
Published April 15, 1999
Citation Information: J Clin Invest. 1999;103(8):1179-1190. https://doi.org/10.1172/JCI1918.
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Article

A potential role for protease nexin 1 overexpression in the pathogenesis of scleroderma

Abstract

Scleroderma currently affects approximately 75,000–100,000 individuals in the United States. Fibroblasts isolated from lesional skin of scleroderma patients overexpress collagens and other matrix components, and this abnormality is maintained for multiple passages in culture. To understand the molecular basis for matrix gene overexpression, we performed a differential display comparison of fibroblasts from clinically lesional and nonlesional scleroderma skin. The results suggested that protease nexin 1 (PN1), a protease inhibitor, is overexpressed in scleroderma fibroblasts. Northern blot verification showed that lesional and nonlesional scleroderma fibroblasts had three- to five-fold increased levels of PN1 mRNA compared with healthy fibroblasts. Western analysis showed that scleroderma fibroblasts also secreted more PN1. In situ hybridization of skin biopsy specimens demonstrated PN1 expression in the dermis of four out of six scleroderma patients but no PN1 expression in the dermis of six healthy volunteers. Transient or stable overexpression of PN1 in mouse 3T3 fibroblasts increased collagen promoter activity or endogenous collagen transcript levels, respectively. PN1 mutagenized at its active site and antisense PN1 both failed to increase collagen promoter activity. These results suggest that overexpression of enzymatically active PN1 may play a pathogenic role in the development of the scleroderma phenotype.

Authors

David Strehlow, Ante Jelaska, Karen Strehlow, Joseph H. Korn

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Figure 1

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Lanes 1–8 and 9–16 show an example of differential display of RNA from s...
Lanes 1–8 and 9–16 show an example of differential display of RNA from scleroderma biopsy fibroblasts using two primer pairs. The 5′ primer for lanes 1–8 was RDS3, 5′-GAATTCACCAGAC; the 5′ primer for lanes 9–16 was RDS4, 5′-GAATTCGCTCACT; the 3′ primer for all samples shown is the same: HT11A, 5′-AAGCTTTTTTTTTTTA. Duplicate samples of RNA from nonlesional (N) and lesional (L) fibroblasts are shown in the designated lanes. Asterisks represent differential display bands, which are uniformly represented in nonlesional and lesional tissue from a given patient, but which differ between patients. The PN1 band is shown at the arrow and is amplified more in the nonlesional tissue (lanes 13 and 14) than in the lesional (lanes 15 and 16). White puncture marks in the x-ray film, used to define the band on the gel before cloning, are visible for PN1. Fortuitously, PN1 was amplified in all lanes where the 3′ primer HT11A was used, regardless of the sequence of the 5′ primer. We subsequently showed that the band was amplified by using the primer HT11A at both ends of the template (data not shown).