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BMC Cell Biology, 2009
Background: Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the trans-acting proteins AU rich binding factor 1 (AUF1), Upstream of Nras (Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay.
Journal of Clinical Investigation, 2009
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.
The FASEB Journal, 2008
Serum calcium and phosphate concentrations and experimental chronic kidney failure control parathyroid hormone (PTH) gene expression posttranscriptionally through regulated binding of the transacting proteins AUF1 and upstream of N-ras (Unr) to an AU-rich element (ARE) in PTH mRNA 3-untranslated region (3UTR). We show that the mRNA decay promoting K-homology splicing regulator protein (KSRP) binds to PTH mRNA in intact parathyroid glands and in transfected cells. This binding is decreased in glands from calcium-depleted or experimental chronic kidney failure rats in which PTH mRNA is more stable compared to parathyroid glands from control and phosphorus-depleted rats in which PTH mRNA is less stable. PTH mRNA decay depends on the KSRP-recruited exosome in parathyroid extracts. In transfected cells, KSRP overexpression and knockdown experiments show that KSRP decreases PTH mRNA stability and steady-state levels through the PTH mRNA ARE. Overexpression of isoform p45 of the PTH mRNA stabilizing protein AUF1 blocks KSRP-PTH mRNA binding and partially prevents the KSRP mediated decrease in PTH mRNA levels. Therefore, calcium or phosphorus depletion, as well as chronic kidney failure, regulate the interaction of KSRP and AUF1 with PTH mRNA and its half-life. Our data indicate a novel role for KSRP in PTH gene expression.-Nechama, M., Ben-Dov, I. Z., Briata, P., Gherzi, R., Naveh-Many, T. The mRNA decay promoting factor K-homology splicing regulator protein post-transcriptionally determines parathyroid hormone (PTH) mRNA levels. FASEB J. 22, 3458 -3468 (2008)
Biochemistry, 1994
Parathyroid hormone-related protein (PTHrP) is expressed by malignant tumors and leads to the syndrome of humoral hypercalcemia of malignancy. It is also expressed by a wide variety of nonmalignant tissues, in which it appears to play distinct paracrine and/or autocrine roles. The human PTHrP gene encodes three cDNA-predicted initial translational products of 139, 141, and 173 amino acids. Most human cell lines contain mRNAs encoding all three PTHrP isoforms. The physiological rationale for the existence of these three highly similar transcripts is unknown. In order to determine whether the protein products derived from these three transcripts differ, we transfected Chinese hamster ovary (CHO) cells and rat insulinoma (RIN) cells individually with cDNAs encoding human PTHrP( 1-139), PTHrP( 1-141), and PTHrP( 1-1 73). Cell extracts and conditioned medium were then chromatographed using reversedphase HPLC and analyzed using region-specific PTHrP immunoassays. As we had previously observed in SKRC-1 (renal cell carcinoma) and R I N ( 1-141) cells, multiple amino-terminal PTHrP species as well as a separate midregion PTHrP species were identified in all six cell lines. In addition, both C H O and R I N cell lines transfected with the PTHrP( 1-1 39) construct contained a previously unrecognized carboxyterminal fragment that reacted with a PTHrP antiserum. This carboxy-terminal fragment was physically distinct from the midregion fragment discovered earlier and was also present in conditioned medium, indicating that it is a secretory form, rather than a biosynthetic intermediate or a degradation product. Surprisingly, R I N and C H O cells transfected with PTHrP( 1-141) or -( 1-173) contained little of this carboxy-terminal fragment, suggesting that isoform-specific protein processing exists for PTHrP. In addition, while R I N cells produced a single predominant amino-terminal species, C H O cells contained two, approximately equimolar, amino-terminal species, indicating the existence of cell-specific protein processing. These studies indicate that the posttranslational processing of PTHrP is highly complex. Specifically, (a) multiple amino-terminal PTHrP secretory forms, as well as a midregion form, are generated by cell lines containing each of the three PTHrP transcripts; (b) the Arg3' cleavage that generates the midregion fragment occurs in the Golgi apparatus, as both constitutive and regulated secretory cell types are capable of performing this cleavage; (c) a previously unrecognized carboxy-terminal fragment of PTHrP is secreted; and (d) processing of PTHrP appears to be both isoform-and cell-specific. Complete structural determination of each of these fragments is critical to understanding PTHrP physiology and pathophysiology.
Endocrinology, 2006
The type 1 PTH/PTH-related peptide receptor (PTH1R) is a class B G protein-coupled receptor that demonstrates immunoreactivity in the nucleus as well as cytoplasm of target cells. Our previous studies on the PTH1R have shown that it associates with the importin family of transport regulatory proteins. To investigate the role of the importins in PTH1R nuclear import, we used small interfering (si)RNA technology to knock down the expression of importin- in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin- siRNA-treated cells demonstrated a decrease in nuclear localization of the PTH1R in comparison with control cells. Under normal culture conditions, PTH1R is present in both the nucleus and cytoplasm of cells. Serum starvation favors nuclear localization of PTH1R, whereas returning cells to serum or treatment with PTH-related peptide induced its cytoplasmic localization. To address the nuclear export of PTH1R, interactions between PTH1R and chromosomal region maintenance 1 (CRM1) were investigated. PTH1R and CRM1 coimmunoprecipitated from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, PTH1R accumulated in the nucleus. Taken together, our studies show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-␣/ and CRM1. (Endocrinology 148:
Journal of Biological Chemistry, 1998
Parathyroid hormone (PTH) regulates serum calcium and phosphate levels, which, in turn, regulate PTH secretion and mRNA levels. PTH mRNA levels are markedly increased in rats fed low calcium diets and decreased after low phosphate diets, and this effect is posttranscriptional. Protein-PTH mRNA binding studies, with parathyroid cytosolic proteins, showed three protein-RNA bands. This binding was to the 3-untranslated region (UTR) of the PTH mRNA and was dependent upon the terminal 60 nucleotides. Parathyroid proteins from hypocalcemic rats showed increased binding, and proteins from hypophosphatemic rats decreased binding, correlating with PTH mRNA levels. There is no parathyroid cell line; however, a functional role was provided by an in vitro degradation assay. Parathyroid proteins from control rats incubated with a PTH mRNA probe led to an intact transcript for 40 min; the transcript was intact with hypocalcemic proteins for 180 min and with hypophosphatemic proteins only for 5 min. A PTH mRNA probe without the 3-UTR, or just the terminal 60 nucleotides, incubated with hypophosphatemic proteins, showed no degradation at all, indicating that the sequences in the 3-UTR determine PTH mRNA degradation. Hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally. This correlates with binding of proteins to the PTH mRNA 3-UTR, which determines its stability.
Molecular basis of parathyroid hormone receptor signaling and trafficking: a family B GPCR paradigm
Cellular and Molecular Life Sciences, 2011
The parathyroid hormone (PTH) receptor type 1 (PTHR), a G protein-coupled receptor (GPCR), transmits signals to two hormone systems-PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrineto regulate different biological processes. PTHR responds to these hormonal stimuli by activating heterotrimeric G proteins, such as G S that stimulates cAMP production. It was thought that the PTHR, as for all other GPCRs, is only active and signals through G proteins on the cell membrane, and internalizes into a cell to be desensitized and eventually degraded or recycled. Recent studies with cultured cell and animal models reveal a new pathway that involves sustained cAMP signaling from intracellular domains. Not only do these studies challenge the paradigm that cAMP production triggered by activated GPCRs originates exclusively at the cell membrane but they also advance a comprehensive model to account for the functional differences between PTH and PTHrP acting through the same receptor.
Parathyroid Hormone-Related Protein: An Update
The Journal of Clinical Endocrinology & Metabolism, 2012
PTHrP was identified as a cause of hypercalcemia in cancer patients 25 yr ago. In the intervening years, we have learned that PTHrP and PTH are encoded by related genes that are part of a larger "PTH gene family." This evolutionary relationship permits them to bind to the same type 1 PTH/ PTHrP receptor, which explains why humoral hypercalcemia of malignancy resembles hyperparathyroidism. This review will outline basic facts about PTHrP biology and its normal physiological functions, with an emphasis on new findings of the past 5-10 yr. The medical and research communities first became aware of PTHrP because of its involvement in a common paraneoplastic syndrome. Now, research into the basic biology of PTHrP has suggested previously unrecognized connections to a variety of disease states such as osteoporosis, osteoarthritis, and breast cancer and has highlighted how PTHrP itself might be used in therapy for osteoporosis and diabetes. Therefore, the story of this remarkable protein is a paradigm for translational research, having gone from bedside to bench and now back to bedside.
Journal of Biological Chemistry, 2000
Parathyroid hormone (PTH) mRNA levels are posttranscriptionally increased by hypocalcemia and decreased by hypophosphatemia, and this is mediated by cytosolic proteins binding to the PTH mRNA 3-untranslated region (UTR). The same proteins are also present in other tissues, such as brain, but only in the parathyroid is their binding regulated by calcium and phosphate. The function of the PTH mRNA 3-UTR-binding proteins was studied using an in vitro degradation assay. Competition for the parathyroid-binding proteins by excess unlabeled 3-UTR destabilized the full-length PTH transcript in this assay, indicating that these proteins protect the RNA from RNase activity. The PTH RNA 3-UTR-binding proteins were purified by RNA affinity chromatography of rat brain S-100 extracts. The eluate from the column was enriched in PTH RNA 3-UTR binding activity. Addition of eluate to the in vitro degradation assay with parathyroid protein extracts stabilized the PTH transcript. A major band from the eluate at 50 kDa was sequenced and was identical to AU-rich binding protein (AUF1). Recombinant AUF1 bound the full-length PTH mRNA and the 3-UTR. Added recombinant AUF1 also stabilized the PTH transcript in the in vitro degradation assay. Our results show that AUF1 is a protein that binds to the PTH mRNA 3-UTR and stabilizes the PTH transcript.