Elastase and neutral cathepsin production by human fibroblasts: Effect of culture conditions on synthesis and secretion

David E. Schwartz*, Amy S. Paller, Paula P. Lizak, Roger W. Pearson

*Corresponding author for this work

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23 Scopus citations

Abstract

Fibroblasts from normal adult forearm skin and neonatal foreskin were cultured and examined for their ability to synthesize and secrete elastase and neutral cathepsin. All of the cultures examined produced detectable amounts of elastase using insoluble elastin as substrate. An enzyme was also found that hydrolyzed the synthetic elastin substrate, N-succinyl-(Ala)3-p-nitroanilide, but did not degrade insoluble elastin. In addition, activity against the synthetic cathepsin substrate N-benzoyl-dl-phenylalanine-naphthyl ester was found. Inhibitor profiles indicate that the elastin and N-succinyl-(Ala)3-p-nitroanilide degrading activities are due to metalloproteinases. Degradation of N-benzoyl-dl-phenylalanine-naphthyl ester can be inhibited by phenylmethylsulfonyl fluoride. These proteinases were usually found associated with the cell layer. Although activities of the measured proteinases were detected in all cultures, increased or decreased enzyme activities were not predictably related to passage number or length of serum starvation. Degree of confluence also affected proteinase activities. Separation of the dermal-epidermal junction can be produced by the injection of these proteinases into intact mouse skin.

Original languageEnglish (US)
Pages (from-to)63-68
Number of pages6
JournalJournal of Investigative Dermatology
Volume86
Issue number1
DOIs
StatePublished - Jan 1986

Funding

he integrity of normal dermis is maintained by a bal-ance of synthesis and degradation of extracellular ma-trix proteins, processes largely directed by dermal fibroblasts. Although the synthesis of connective tis-T sue components by fibroblasts has been studied extensively, relatively little attention has been given to the examination of fibroblast proteinases. Extracts of normal dermis and dermis from individuals with cutaneous disorders have been shown to contain proteinases that degrade synthetic trypsin and chymotrypsin substrates and hydrolyze a wide variety of proteins, including casein, hemoglobin, gelatin, and fibrin [1-3[. Fibroblast colbgenase has been studied in cultures of nornral and drug-treated cells [4-10 I and in tissue extracts and cultured fibroblasts from patients with various diseases, especially recessive dystrophic epidermolysis bullosa [1 I j, malignant cutaneous tumors [12-1 6], periodontal di sease [1 7, 18], and rheuma toid arthritis [191. Dermal fibroblasts in culture have also been shown to produce an elastase [20-221 and a proteinase that cleaves a synthetic substrate for elastase, N-succinyl-(alanine)J-p-nitroanilide (SANA), but not insoluble elastin [211. We have recently Manuscript recei ved February 8. 1985; accepted for public:Hiorr Jul y 24, 1985. This work w~s supported by NIH Grarrt H23-EY-04U I7 (DES). Grarrt 1394 from the Courrcil for Tob~cco Hcsc~rclr lrrc., U.S.A. (DES), National Arthritis Foundation Fellowship (DES), and by OCLS. lhrsh-Prcsbytcrian-St. Luke's Medical Center. Hcprint requests to: David E. Schwartz, I' h. D., Departnrerrt of l3iochemistry, Rush-Presbyterian-St. Luke's Medical Center, 1753 West Congress Parkway, Chicago, Illinois 60612. Abbreviations: BPN E: bcnzoy i-DL-phenylalarrirrc naphthyl ester DMEM: Dulbccco's rnodifrcd Eagle's medium EGT A: ethylencglyco l-bis(,B-anr in octh yl cthcr)-N, N '-tctraacetic acid PMSF: phenylmcthylsulfonyl Auoridc SANA: N-succinyi-(Aia)J-p-nitroanilide TNC: 0.25 M Tris-HCI, pH 7.3, and 0.025 M CaCI~ are due to metalloproteinases. Degradation of N-benzoylDL-phenylalanine-naphthyl ester can be inhibited by phenylmethylsulfonyl fluoride. These proteinases were usually found associated with the cell layer. Although activities of the measured proteinases were detected in all cultures, increased or d ecreased enzyme activities w ere not predictabl y related to passage number or length of serum starvation. Degree of confluence also affected proteinase activities. Separation of the dermal-epidermal junction can be produced by the injection of these proteinascs into intact mouse skin.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

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