RESUMEN

Teriparatide, an osteoanabolic agent, is a biosynthetic analogue of the 1-34 amino acids of human parathyroid hormone (PTH) used for the treatment of osteoporosis. It is typically well-tolerated; common side effects include headaches, arthralgias, nausea, and dizziness. In this report, we present a case of gynecomastia occurring shortly after initiating teriparatide therapy, associated with nipple sensitivity and breast tenderness. Secondary workup for various causes of gynecomastia was unremarkable. Finally, a decision was made to discontinue teriparatide due to the patient's concerns. The nipple sensitivity started improving shortly afterward, with complete resolution of gynecomastia 4 months later. Although this unusual side effect has been reported as a possibility in postmarketing studies, a chronological report on the occurrence of teriparatide-induced gynecomastia and its complete resolution after discontinuing teriparatide has not yet been published in the literature.

RESUMEN

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells. AIM: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models. METHODS: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 µg/kg) in 100 µL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05. RESULTS: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05). CONCLUSION: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Asunto(s)

RESUMEN

The canonical model for G protein-coupled receptors (GPCRs) activation assumes that stimulation of heterotrimeric G protein signaling upon ligand binding occurs solely at the cell surface and that duration of the stimulation is transient to prevent overstimulation. In this model, GPCR signaling is turned-off by receptor phosphorylation via GPCR kinases (GRKs) and subsequent recruitment of ß-arrestins, resulting in receptor internalization into endosomes. Internalized receptors can then recycle back to the cell surface or be trafficked to lysosomes for degradation. However, over the last decade, this model has been extended by discovering that some internalized GPCRs continue to signal via G proteins from endosomes. This is the case for the parathyroid hormone (PTH) type 1 receptor (PTHR), which engages on sustained cAMP signaling from endosomes upon PTH stimulation. Accumulative evidence shows that the location of signaling has an impact on the physiological effects of GPCR signaling. This mini-review discusses recent insights into the mechanisms of PTHR endosomal signaling and its physiological impact.

Asunto(s)

RESUMEN

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Asunto(s)

RESUMEN

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained Gs signaling at endosomes. In addition to Gs, PTHR1 activates Gq signaling; yet, in contrast to the PTHR1-Gs pathway, the spatiotemporal dynamics of the Gq branch of PTHR1 signaling and its relationship to Gs signaling remain largely ill defined. Recognizing that a downstream consequence of Gq signaling is the activation of phospholipase D (PLD) enzymes, we leverage activity-based, bioorthogonal imaging tools for PLD signaling to visualize and quantify the Gq branch of PTHR1 signaling. We establish that PTHR1-Gq signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein Gq and Gs compete for ligand-bound receptors at the PM and more broadly highlight the utility of bioorthogonal tools for imaging PLDs as probes to visualize GPCR-Gq signaling.

Asunto(s)

RESUMEN

El hiperparatiroidismo secundario es uno de los componentes integrales de las alteraciones del metabolismo óseo-mineral en la enfermedad renal crónica (ERC) o complejo chronic kidney disease-mineral bone disorder. Se ha demostrado que en el desarrollo y progresión del hiperparatiroidismo secundario intervienen muchos factores, estrechamente interrelacionados, pero la presencia e importancia de la hiporrespuesta (o resistencia) a la acción de la hormona paratiroidea (PTH) es poco comprendida. En esta revisión analizaremos sus antecedentes, factores que intervienen, así como alguno de los mecanismos moleculares que podrían explicarla. La presencia de resistencia a la acción biológica de la PTH no es única en la ERC, ya que también se presenta para otras hormonas, habiéndose incluso usado el término de «uremia como una enfermedad de receptores». Esta hiporrespuesta a la PTH tiene importantes implicaciones clínicas, dado que no solo permite explicar parte de la patogenia progresiva de la hipersecreción de PTH e hiperplasia paratiroidea, sino también la creciente prevalencia de enfermedad ósea adinámica en la población con ERC. De este modo, subrayamos la importancia de controlar, sin normalizar completamente, los niveles de PTH en los distintos estadios de ERC, dado que un cierto incremento de sus niveles supone inicialmente una adaptación clínica. Futuros estudios a nivel molecular sobre la uremia, o la reciente descripción del efecto directo del fosfato sobre la actividad del receptor sensor de calcio como sensor de fosfato, podrían resultar valiosos incluso más allá de explicar la hiporrespuesta a la PTH en la ERC. (AU)

Secondary hyperparathyroidism (SHPT) is an integral component of the chronic kidney disease–mineral and bone disorder (CKD–MBD). Many factors have been associated with the development and progression of SHPT but the presence of skeletal or calcemic resistance to the action of PTH in CKD has often gone unnoticed. The term hyporesponsiveness to PTH is currently preferred and, in this chapter, we will not only review the scientific timeline but also some of the molecular mechanisms behind. Moreover, the presence of resistance to the biological action of PTH is not unique in CKD since resistance to other hormones has also been described (“uremia as a receptor disease”). This hyporesponsiveness carries out important clinical implications since it explains, at least partially, not only the progressive nature of the pathogenesis of CKD-related PTH hypersecretion and parathyroid hyperplasia but also the increasing prevalence of adynamic bone disease in the CKD population. Therefore, we underline the importance of PTH control in all CKD stages, but not aiming to completely normalize PTH levels since a certain degree of SHPT may represent an adaptive clinical response. Future studies at the molecular level, i.e. on uremia, or the recent description of the calcium-sensing receptor as a phosphate sensor, may become of great value beyond their significance to explain just the hyporesponsiveness to PTH in CKD. (AU)

Asunto(s)

RESUMEN

Feeding low dietary cation-anion difference (DCAD) diets is one strategy to prevent milk fever in cows. The mechanism of action, as well as whether the calcium (Ca) supply of such diets combined with this feeding regimen should meet the requirements, is still unclear. Small ruminants are commonly used as models for cows. The goal of the present study was to demonstrate basic effects of DCAD against a background of different Ca supplies in a sheep model. Twenty-three castrated male East Friesian milk sheep, aged 11 to 12 mo, were randomly assigned to 4 different feeding groups. The ration of each group was either high (highDCAD) or low in DCAD (lowDCAD) combined with adequate (nCa) or restricted Ca supply (lowCa). At baseline, serum and urine were collected from all sheep and a peripheral quantitative computed tomography of the left metatarsus was performed. After a 14-d adaptation period to the different diets, the experiment started (d 0). Urine, feces, and serum were collected on d 0, 4, 7, 14, and 22, and peripheral quantitative computed tomography was performed on d 0 and 22. On d 22, the sheep were killed and sampled for functional studies. LowDCAD was significantly associated with lower urine pH, higher urinary Ca excretion, higher ionized Ca in blood, and higher serum Ca concentrations. Blood pH and bone parameters did not differ significantly between groups. It is unclear from which compartment the high amounts of Ca excreted with urine in the lowDCAD groups originated. Interestingly, lowDCAD resulted in higher renal mRNA abundance of parathyroid hormone receptor but unaffected mRNA abundance of Ca transporters. As neither renal abundance of these transporters nor Ca excretion were influenced by dietary Ca supply, our results support the hypothesis that increased urinary Ca observed with low DCAD diets represents a loss rather than an excretion of surplus Ca.

Asunto(s)

RESUMEN

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.

Asunto(s)

RESUMEN

Secondary hyperparathyroidism (SHPT) is an integral component of the chronic kidney disease-mineral and bone disorder (CKD-MBD). Many factors have been associated with the development and progression of SHPT but the presence of skeletal or calcemic resistance to the action of PTH in CKD has often gone unnoticed. The term hyporesponsiveness to PTH is currently preferred and, in this chapter, we will not only review the scientific timeline but also some of the molecular mechanisms behind. Moreover, the presence of resistance to the biological action of PTH is not unique in CKD since resistance to other hormones has also been described ("uremia as a receptor disease"). This hyporesponsiveness carries out important clinical implications since it explains, at least partially, not only the progressive nature of the pathogenesis of CKD-related PTH hypersecretion and parathyroid hyperplasia but also the increasing prevalence of adynamic bone disease in the CKD population. Therefore, we underline the importance of PTH control in all CKD stages, but not aiming to completely normalize PTH levels since a certain degree of SHPT may represent an adaptive clinical response. Future studies at the molecular level, i.e. on uremia or the recent description of the calcium-sensing receptor as a phosphate sensor, may become of great value beyond their significance to explain just the hyporesponsiveness to PTH in CKD.

Asunto(s)

RESUMEN

Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated Gs/adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/Gs signaling regulated by Tctex-1.

Asunto(s)

RESUMEN

Recent studies demonstrate that parathyroid hormone (PTH) not only maintains mineral homeostasis through targeting the kidneys and bone, but also exerts its effects on other organs. For instance, PTH induces urate accumulation through inhibiting the expression of the ABCG2 in both the intestine and the kidney. In addition, PTH downregulates the expression of cytochrome P450 (CYP) 3A, a major enzyme for drug metabolism in both the intestine and liver, resulting in the increase of substrate drug exposure. These functions of PTH are mediated through the PTH receptor (PTHR) signaling. Since PTHR exists in various organs, PTH may regulate other, still unspecified transporters or enzymes in the organs that express PTHR.

Asunto(s)

RESUMEN

G protein-coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, ß-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires ß-arrestin and does not display a preference for ß-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit ß-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed ß-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.

Asunto(s)

RESUMEN

Osteoporosis is a progressive bone disorder characterised by imbalance between bone building (anabolism) and resorption (catabolism). Most therapeutics target inhibition of osteoclast-mediated bone resorption, but more recent attention in early drug discovery has focussed on anabolic targets in osteoblasts or their precursors. Two marketed agents that display anabolic properties, strontium ranelate and teriparatide, mediate their actions via the G protein-coupled calcium-sensing and parathyroid hormone-1 receptors, respectively. This review explores their activity, the potential for improved therapeutics targeting these receptors and other putative anabolic GPCR targets, including Smoothened, Wnt/Frizzled, relaxin family peptide, adenosine, cannabinoid, prostaglandin and sphingosine-1-phosphate receptors.

Asunto(s)

RESUMEN

BACKGROUND: As an invasive malignant tumor, osteosarcoma (OS) has high mortality. Parathyroid hormone receptor 1 (PTHR1) contributes to maintaining proliferation and undifferentiated state of OS. This study is designed to reveal the action mechanisms of PTHR1 in OS. METHODS: Microarray dataset GSE46861, which included six PTHR1 knockdown OS samples and six control OS samples, was obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified and then performed with enrichment analysis separately using the limma package and DAVID online tool. Then, protein-protein interaction (PPI) network and module analyses were conducted using Cytoscape software. Using the WebGestalt tool, microRNAs (miRNAs) were predicted for the DEGs involved in the PPI network. Following this, transcription factors (TFs) were predicted and an integrated network was constructed by Cytoscape software. RESULTS: There were 871 DEGs in the PTHR1 knockdown OS samples compared with the control OS samples. Besides, upregulated ZFPM2 was involved in the miRNA-DEG regulatory network. Moreover, TF LEF1 was predicted for the miRNA-DEG regulatory network of the downregulated genes. In addition, LEF1, NR4A2, HAS2, and RHOC had higher degrees in the integrated network. CONCLUSIONS: ZFPM2, LEF1, NR4A2, HAS2, and RHOC might be potential targets of PTHR1 in OS.

Asunto(s)

RESUMEN

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

Asunto(s)

RESUMEN

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry and bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.

Asunto(s)

RESUMEN

Parathyroid hormone-related protein (PTHrP) inhibits proliferation of several lung cancer cell lines, but the signaling mechanism has not been established. This study tested the hypotheses that growth inhibition is mediated through the PTHrP receptor, PTH1R, and that the process is modified by ERK activation. PTHrP-positive and negative clones of H1944 lung adenocarcinoma cells underwent stable PTH1R knockdown with lentiviral shRNA or transient transfection with ERK1 and ERK2 siRNA. Alternatively, cells were treated with 8-CPT cAMP, 8-CPT 2'-O-methyl cAMP, and N-6-phenyl cAMP analogs. H1944 cells expressing ectopic PTHrP showed 20-40% decrease in proliferation compared to the PTHrP-negative cells in the presence of normal levels of PTH1R (P < 0.01). PTH1R knockdown eliminated this difference and increased cell proliferation regardless of PTHrP status. The three cAMP analogs each inhibited proliferation over 5 days by 30-40%. ERK2 knockdown inhibited proliferation of PTHrP-positive cells alone and in combination with ERK1 knockdown. The growth inhibition mediated by cAMP analogs was unaffected by ERK1 knockdown. In conclusion, ectopic expression of PTHrP 1-87 inhibits H1944 cell proliferation. PTH1R knockdown blocks this effect and stimulates proliferation, indicating that the ligand exerts anti-mitogenic effects. cAMP, the second messenger for PTH1R also inhibits proliferation and activates ERK. PTHrP growth inhibition may be opposed by concomitant ERK activation.

RESUMEN

The presence of the parathyroid hormones in vertebrates, including PTH, PTH-related peptide (PTHrP), and tuberoinfundibular peptide of 39 residues (TIP39), has been proposed to be the result of two rounds of whole genome duplication in the beginning of vertebrate diversification. Bioinformatics analyses, in particular chromosomal synteny study and the characterization of the PTH ligands and their receptors from various vertebrate species, provide evidence that strongly supports this hypothesis. In this mini-review, we summarize recent advances in studies regarding the molecular evolution and physiology of the PTH ligands and their receptors, with particular focus on non-mammalian vertebrates. In summary, the PTH family of peptides probably predates early vertebrate evolution, indicating a more ancient existence as well as a function of these peptides in invertebrates.

RESUMEN

Inappropriate aldosterone and parathyroid hormone (PTH) secretion is strongly linked with development and progression of cardiovascular (CV) disease. Accumulating evidence suggests a bidirectional interplay between parathyroid hormone and aldosterone. This interaction may lead to a disproportionally increased risk of CV damage, metabolic and bone diseases. This review focuses on mechanisms underlying the mutual interplay between aldosterone and PTH as well as their potential impact on CV, metabolic and bone health. PTH stimulates aldosterone secretion by increasing the calcium concentration in the cells of the adrenal zona glomerulosa as a result of binding to the PTH/PTH-rP receptor and indirectly by potentiating angiotensin 2 induced effects. This may explain why after parathyroidectomy lower aldosterone levels are seen in parallel with improved cardiovascular outcomes. Aldosterone mediated effects are inappropriately pronounced in conditions such as chronic heart failure, excess dietary salt intake (relative aldosterone excess) and primary aldosteronism. PTH is increased as a result of (1) the MR (mineralocorticoid receptor) mediated calciuretic and magnesiuretic effects with a trend of hypocalcemia and hypomagnesemia; the resulting secondary hyperparathyroidism causes myocardial fibrosis and disturbed bone metabolism; and (2) direct effects of aldosterone on parathyroid cells via binding to the MR. This adverse sequence is interrupted by mineralocorticoid receptor blockade and adrenalectomy. Hyperaldosteronism due to klotho deficiency results in vascular calcification, which can be mitigated by spironolactone treatment. In view of the documented reciprocal interaction between aldosterone and PTH as well as the potentially ensuing target organ damage, studies are needed to evaluate diagnostic and therapeutic strategies to address this increasingly recognized pathophysiological phenomenon.

Asunto(s)

RESUMEN

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009-2012). Of the 215 patients [140 males and 75 females (male/female ratio=1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n=20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n=8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.

Asunto(s)