Ab1802

Aberrant proteolytic digestion of biglycan and decorin by saliva and exocrine gland lysates from the NOD mouse model for autoimmune exocrinopathy

S. Yamachika¹, J. Brayer¹, G.E. Oxford¹, A.B. Peck^3,4, M.G. Humphreys-Beher^1,3

¹Department of Oral Biology, ²Department of Pathology and Laboratory Science, and the ³Center for Orphaned Autoimmune Disease, University of Florida, Gainesville, Florida, USA.

ABSTRACT
Objective
The protein components of the extracellular matrix (ECM) are responsible for driving tissue morphogenesis, the development of differentiated function, and the sequestration of biologically active molecules such as growth factors in close proximity to tissue and organ cells. Recent reports indicate that saliva and exocrine tissue lysates from Sjögren's syndrome patients and the non-obese diabetic (NOD) mouse model for autoimmune exocrinopathy demonstrate elevated levels of specific enzymes that degrade the ECM, especially the matrix metalloproteinases (MMPs). To determine if elevated levels of MMPs could be important in exocrine tissue destruction, we examined proteolytic activity against two ECM proteoglycans, decorin and biglycan.

Methods
Purified recombinant human core protein for decorin or biglycan was incubated with saliva or gland lysates from either control BALB/c or NOD mice. Degraded proteoglycan products were estimated by Western blotting analysis using anti-decorin or anti-biglycan monospecific polyclonal antibodies. The levels of TGFb protein were measured by ELISA.

Results
Proteolytic activity for decorin and biglycan was not observed in the saliva and salivary gland lysates collected from C57BL/6 or BALB/c mice used as normal controls. In contrast, both proteoglycans were degraded by saliva and exocrine gland lysates from NOD mice and the congenic partner strains NOD-scid and NOD.B10.H2^b. This proteolytic activity for proteoglycans was inhibited by the MMP inhibitors, EDTA, GM6001 and 1,10-phenanthroline. Protein steady state levels for TGFb were increased in the saliva and gland lysates from 20-week old NOD strains, as compared to BALB/c mice and NOD treated with the MMP inhibitor GM6001. With the inhibition of MMP activity, TGFb levels declined in saliva and gland lysates.

Conclusion
Proteolytic degradation of the ECM molecules decorin and biglycan is elevated in the exocrine tissues of the NOD mouse model for Sjögren's syndrome. Furthermore, the proteolysis of small leucine-rich proteoglycans correlates with the presence of elevated levels of TGFb in gland lysates and saliva.

Key words
Extracellular matrix proteins, Sjögren's syndrome, proteases, exocrine glands.

This work was supported by NIH/NIDR grant DE 10515 and by an administrative supplement from the Office for Research in Women's Health Policy to Dr. Humphreys-Beher. Dr. Peck was supported by a Juvenile Diabetes Foundation International grant (I96091). Mr. Brayer and Dr. Oxford were supported by NIDR training grant DE 00072.

Please address correspondence and reprint requests to: Dr. Michael G. Humphreys-Beher, Department of Oral Biology, P.O. Box 100424, University of Florida, Gainesville, FL 32610, USA.
E-mail: h-beher@dental.ufl.edu