Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Abstract

Preproparathyroid hormone (prepro-PTH) is one of the proteins abundantly synthesized by parathyroid chief cells; yet under normal growth conditions, little or no prepro-PTH can be detected in these cells. Although this may be attributed to effective cotranslational translocation and proteolytic processing, proteasome-mediated degradation of PTH precursors may be important in the regulation of the levels of these precursors and hence PTH secretion. The effects of N-acetyl-Leu-Leunorleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-IleGlu(t-butyl)-Ala-leucinal (proteasome inhibitor I), and lactacystin on the biosynthesis and secretion of PTH were examined in dispersed bovine parathyroid cells. We demonstrate that treatment of these cells with proteasome inhibitors caused the accumulation of prepro-PTH and pro-PTH. Compared with mock-treated cells, the processing of pro-PTH to PTH was delayed, and the secretion of intact PTH decreased in proteasome inhibitor-treated cells. Relieving the inhibition of the proteasome by chasing MG132-treated cells in medium without the inhibitor led to the rapid disappearance of the accumulated prepro-PTH, and the rate of PTH secretion was restored to levels comparable to those in mocktreated cells. Furthermore, overexpression of the Hsp70 family of molecular chaperones was observed in proteasome inhibitor-treated cells, and we show that PTH/PTH precursors interact with these molecular chaperones. These data suggest the involvement of parathyroid cell proteasomes in the quality control of PTH biosynthesis.The calcium concentration in mammalian body fluids is tightly regulated predominantly by the actions of parathyroid hormone (PTH) 1 on bone, kidney, and intestine (1-4 ] o ) secretion of PTH. However, the signaling pathway(s) leading to the rapid and specific changes in PTH secretion remains largely unknown.The primary precursor prepro-PTH is translocated into the endoplasmic reticulum (ER) and cleaved to pro-PTH within 1 min. Pro-PTH then transits the ER and attains the trans-Golgi network within 20 min, where it is cleaved to mature PTH. Depending on the needs and predominantly on [Ca 2ϩ ] o , mature PTH is packaged into either secretory granules for exocytosis or storage granules. Secretion of de novo synthesized PTH is believed to occur within 30 min of prepro-PTH formation (6) and constitutes the bulk of secreted PTH (7-9). At the posttranscriptional level, acute changes in [Ca 2ϩ ] o (Ͻ24 h) do not significantly affect the rate of PTH biosynthesis. However, sustained or chronic hypocalcemia and hypercalcemia affect the stability and hence the levels of PTH mRNA (10 -14, 44).Intracellular proteolysis of mature bioactive PTH has been reported to be one of the mechanisms by which parathyroid cells regulate the amount of hormone available for secretion in response to changes in [Ca 2ϩ ] o (15,16). This PTH metabolism is now known to be mediated by calpains (17) and cathepsins B and D (18 -21). Inhibition of these PTH-degrading ac...

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