Abstract
We purified from porcine pancreas a hypocalcemic peptide clearly distinguishable from other pancreatic osteotropic factors such as amylin, calcitonin, and glucagon. Porcine pancreas was processed by acetone extraction, anion exchange chromatography, isoelectric focusing, and reverse-phase high performance liquid chromatography. Fractions were assayed for their inhibitory effects on bone resorption in vitro. Amino acid sequence of a homogeneous 28-kDa protein revealed 92% homology to a human elastase IIIB in the N terminus. Recombinant human elastase IIIB (rhEIIIB) inhibited bone resorption in organ culture stimulated by 1,25-dihydroxyvitamin D3 at concentrations as low as 75 ng/ml. Antibodies to rhEIIIB recognized purified pancreatic factor in Western blots and blocked its inhibitory effect on bone resorption. This antiresorptive activity was abolished by phenylmethylsulfonyl fluoride, suggesting the importance of elastase proteolytic activity for inhibition of bone resorption. In vivo, rhEIIIB and purified pancreatic factor significantly decreased recombinant human interleukin-1alpha-induced hypercalcemia. In conclusion, a novel naturally occurring inhibitor of bone resorption and calcium-lowering peptide has been identified in porcine pancreas. Because this pancreatic peptide has systemic effects on bone resorption and blood ionized calcium at low concentrations, it may represent a physiological regulator of normal bone remodeling and calcium homeostasis.
Highlights
We have found that systemic administration of extracts of the porcine pancreas (PPE)1 that have been prepared according to the methods described by Takaoka et al [1] reproducibly decrease blood ionized calcium (Ca2ϩ) concentrations in normal mice and inhibit bone resorption, which is stimulated by parathyroid hormone (PTH), interleukin-1␣, prostaglandin E2, tumor necrosis factor, transforming growth factor ␣, and 1,25-dihydroxyvitamin D3 (1,25D3) in organ cul
We have found that this peptide shares about 90% amino acid homology with a serine protease in the elastase family
PPE was purified to homogeneity by successive steps including anion exchange chromatography on DE-52 and DEAESephacel, isoelectric focusing, and reverse-phase high performance liquid chromatography (HPLC) on C18 column
Summary
We have found that systemic administration of extracts of the porcine pancreas (PPE) that have been prepared according to the methods described by Takaoka et al [1] reproducibly decrease blood ionized calcium (Ca2ϩ) concentrations in normal mice and inhibit bone resorption, which is stimulated by parathyroid hormone (PTH), interleukin-1␣, prostaglandin E2, tumor necrosis factor, transforming growth factor ␣, and 1,25-dihydroxyvitamin D3 (1,25D3) in organ cul-. We have found that this peptide shares about 90% amino acid homology with a serine protease in the elastase family (pancreatic elastase IIIB isozyme). Both native pancreatic and rhEIIIB inhibited bone resorption and lowered blood ionized calcium in a dosedependent manner. Antibodies to rhEIIIB recognized native pancreatic and rhEIIIB in Western blots and neutralized bone resorption inhibitory activity of both. These data suggest that the pancreas might play a role as a bone/calcium metabolism-regulating organ by the production of peptides with proteolytic activity
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