The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism
Morris Nechama, … , Justin Silver, Tally Naveh-Many
Morris Nechama, … , Justin Silver, Tally Naveh-Many
Published September 21, 2009
Citation Information: J Clin Invest. 2009;119(10):3102-3114. https://doi.org/10.1172/JCI39522.
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Research Article

The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism

Abstract

Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3′ UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP–PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1–/– mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.

Authors

Morris Nechama, Takafumi Uchida, Irit Mor Yosef-Levi, Justin Silver, Tally Naveh-Many

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Figure 6

Identification of KSRP phosphorylation at S181 and its role in KSRP-Pin1 interaction and PTH mRNA regulation.

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Identification of KSRP phosphorylation at S181 and its role in KSRP-Pin1...
(A) Mass spectrometry. MS spectra of tryptic mixture of immunoprecipitated FLAG-KSRP from transfected cells. The peak at 677.8555 m/z was assigned as the unphosphorylated form of amino acids 177–197 (left), and the peak at 717.8390 m/z the phosphorylated form of the peptide (right). Asterisk indicates serine phosphorylation. The P probability of the assignment was equal to 7.51E-06. Sequest X score was equal to 3.25. (B) KSRP-Pin1 interaction. HEK293 cells were cotransfected with either FLAG-KSRP or FLAG-KSRP S181A and GFP-Pin1 or control plasmid. Extracts were immunoprecipitated with either anti-Pin1 or IgG. The pellets and input were immunoblotted as indicated. (C) KSRP dephosphorylation. Cells were cotransfected with FLAG-KSRP and GFP-Pin1 or control plasmid. Extracts were immunoprecipitated with anti-FLAG or IgG and immunoblotted with anti–phosphor-serine or anti-FLAG antibody. (D–H) Cells were cotransfected with the PTH plasmid or control and either FLAG-KSRP or FLAG-KSRP S181A. (D) Immunoblots showing equal amounts of KSRP. (E) Northern blots of PTH mRNA. The lanes were run on the same gel but were noncontiguous (white line). (F) qRT-PCR for PTH mRNA as in E. *Compared with control; **compared with KSRP. (G) IVDA using cell extracts and full-length human PTH mRNA (top) or PTH mRNA without the ARE (bottom). (H) Quantification of IVDA results in 3 experiments; mean ± SEM of time 0; KSRP compared with both control and S181A; *P < 0.05. (I) Cells were cotransfected with the PTH and KSRP or S181A plasmids with and without juglone. RNA was analyzed by qRT-PCR for PTH mRNA; *P < 0.05 (n = 3) compared with cells expressing KSRP and not treated with juglone.