Prostate Specific Antigen Cleaves Parathyroid Hormone-Related Protein in the PTH-like Domain: Inactivation of PTHrP-Stimulated cAMP Accumulation in Mouse Osteoblasts

Abstract

PURPOSE: To determine whether parathyroid hormone-related protein (PTHrP) is a substrate of prostate-specific antigen (PSA) and how the biological activity of PTHrP may be altered by cleavage with PSA. MATERIALS AND METHODS: Prostate-specific antigen cleavage of recombinant human PTHrP 1-141 was conducted in vitro at 37C and analyzed by SDS-PAGE. Five rounds of automated amino-terminal amino acid sequence analysis were performed on blotted PSA-cleaved PTHrP peptide fragments to determine the PSA cleavage sites. The mouse osteoblast cell line MC3T3-E1 was used to test whether PSA cleavage of PTHrP 1-141 altered its ability to stimulate cAMP production. RESULTS: Prostate-specific antigen was found to specifically cleave PTHrP 1-141 in a time- and dose-dependent manner. Cleavage of PTHrP 1-141 by PSA generated fragments on Coomassie-stained acrylamide gels that migrated with mobilities that corresponded to 19.5, 17, 15 and < 7 kd. The preferred PSA cleavage site of PTHrP 1-141 was determined to be at the carboxyl-terminus of phenylalanine 23, consistent with chymotryptic-like enzymatic activity of PSA. Cleavage of PTHrP by PSA completely abolished the ability of PTHrP to stimulate cAMP production. CONCLUSIONS: Cleavage of PTHrP 1-141 by PSA carboxyl-terminal to phenylalanine 23 represents a unique pattern of PTHrP processing that may be specific to the prostate. Prostate-specific antigen inactivation of the cAMP-inducing activity of PTHrP 1-141 demonstrates that PSA cleavage regulates the biological activity of PTHrP. These results have implications for the role of PTHrP in prostate cancer metastasis to bone and its subsequent regulation of bone remodeling. Study of the biological activities of the PSA-generated PTHrP peptides identified in this study merits further investigation.