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The Role of Protein Kinase C in the Extracellular Ca2+-regulated Secretion of Parathyroid Hormone
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 47
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1384
Keywords [en]
Medical sciences, parathyroid hormone, Ca2+, protein kinase C, calcium sensing receptor, ERK1/2, biosynthesis, secretion
Keywords [sv]
MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-4637ISBN: 91-554-6073-9 (print)OAI: oai:DiVA.org:uu-4637DiVA, id: diva2:165329
Public defence
2004-11-26, C10:301, Uppsala Biomedical Center, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-11-01 Created: 2004-11-01Bibliographically approved
List of papers
1. Biosynthesis and secretion of parathyroid hormone are sensitive to proteasome inhibitors in dispersed bovine parathyroid cells
Open this publication in new window or tab >>Biosynthesis and secretion of parathyroid hormone are sensitive to proteasome inhibitors in dispersed bovine parathyroid cells
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 20, p. 17687-17695Article in journal (Refereed) Published
Abstract [en]

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-Leu-norleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-Ile-Glu(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 mock-treated 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92300 (URN)10.1074/jbc.M108576200 (DOI)11884387 (PubMedID)
External cooperation:
Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2017-12-14Bibliographically approved
2. Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor
Open this publication in new window or tab >>Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor
2004 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 297, no 2, p. 560-573Article in journal (Refereed) Published
Abstract [en]

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92301 (URN)10.1016/j.yexcr.2004.03.039 (DOI)15212956 (PubMedID)
External cooperation:
Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2017-12-14Bibliographically approved
3. Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells over-expressing the calcium sensing receptor
Open this publication in new window or tab >>Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells over-expressing the calcium sensing receptor
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-92302 (URN)
Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2010-01-13Bibliographically approved
4. Physiological activation of PKC-alpha via the calcium sensing receptor is not required for the Ca2+-regulated secretion of parathyroid hormone in dispersed bovine parathyroid cells
Open this publication in new window or tab >>Physiological activation of PKC-alpha via the calcium sensing receptor is not required for the Ca2+-regulated secretion of parathyroid hormone in dispersed bovine parathyroid cells
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-92303 (URN)
Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2011-06-30Bibliographically approved

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