Uncontrolled production or insufficient degradation of lung tissue matrix may alter compliance and gas transfer, causing clinical disorders of ventilation and oxygenation. Certain fibrotic lung diseases demonstrate a high density of mast cells, implicating them in the complex mechanisms of tissue remodeling, an area of investigation focusing on a growing family of matrix-degrading metalloproteinases. The long term objective of this proposal is to determine how resident lung mast cells participate in injury and repair processes during parenchymal inflammation. The preliminary data demonstrate that dog mastocytoma cells secrete and activate their own matrix metalloproteinases (MMPs), supporting the hypothesis that mast cells participate in the regulation of extracellular matrix turnover. Characterization of the proteolytic activity of cell culture supernatants and amino acid analysis of the purified enzyme and its cDNA-predicted peptide sequence suggest secretion of the dog homologue of the human 92-kDa gelatinase. Degranulation studies reveal activation of the gelatinase proenzyme which can be blocked by phenylmethylsulfonyl fluoride (PMSF), further implicating the granule-associated serine proteases, tryptase and chymase, in MMP activation mechanisms. Understanding how mast cells package matrix metalloproteinases may provide insights into interactions with other preformed mediators and in vivo mechanisms of MMP activation.
The specific aims i nclude identification of dog homologues of human MMPs by characterization of substrate specificities and determination of amino acid sequence homology, followed by enzyme purification using sequential chromatographic steps. Activation studies will assess the ability of purified dog mast cell serine proteases to cleave and activate mastocytoma MMPs, and then identify the enzymes responsible for MMP activation in culture. Antibodies will be raised against purified MMPs for use in immunohistochemical studies to determine the cellular localization and tissue distribution of mast cell matrix metalloproteinases. The candidate has completed clinical training in internal medicine, and pulmonary and critical care medicine at UCSF, leading to a strong commitment to academic medicine and an interest in the pathophysiology of lung injury. Research pursued in the laboratory of the sponsor, George Caughey M.D., has focused on mast cell proteases, with the goal of integrating clinical interests in interstitial lung diseases and basic science interests in protein biochemistry and molecular biology. The research training plan includes: an intensive laboratory experience, didactic coursework, weekly seminars and journal clubs, ongoing patient care and clinical teaching, and an advisory committee including the sponsor and experts on matrix metalloproteinases and immunohistopathology, convened to provide scientific guidance and to oversee the applicant's development into an independent investigator in pulmonary research.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL003345-02
Application #
2392548
Study Section
Special Emphasis Panel (ZHL1-CSR-Y (F1))
Project Start
1996-04-01
Project End
2001-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Frank, B T; Rossall, J C; Caughey, G H et al. (2001) Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by alpha-chymase. J Immunol 166:2783-92
Fang, K C (2000) Mesenchymal regulation of alveolar repair in pulmonary fibrosis. Am J Respir Cell Mol Biol 23:142-5
Fang, K C; Wolters, P J; Steinhoff, M et al. (1999) Mast cell expression of gelatinases A and B is regulated by kit ligand and TGF-beta. J Immunol 162:5528-35
Caughey, G H; Raymond, W W; Fang, K C (1998) Porcine origin of human sputum trypsin? Am J Physiol 275:L200-2
Caughey, G H; Blount, J L; Koerber, K L et al. (1997) Cloning and expression of the dog mast cell alpha-chymase gene. J Immunol 159:4367-75
Sommerhoff, C P; Fang, K C; Nadel, J A et al. (1996) Classical second messengers are not involved in proteinase-induced degranulation of airway gland cells. Am J Physiol 271:L796-803