RESUMO
A major challenge in human genetics is the validation of pathogenicity of heterozygous missense variants. This problem is well-illustrated by PROKR2 variants associated with Isolated GnRH Deficiency (IGD). Homozygous, loss of function variants in PROKR2 was initially implicated in autosomal recessive IGD; however, most IGD-associated PROKR2 variants are heterozygous. Moreover, while IGD patient cohorts are enriched for PROKR2 missense variants similar rare variants are also found in normal individuals. To elucidate the pathogenic mechanisms distinguishing IGD-associated PROKR2 variants from rare variants in controls, we assessed 59 variants using three approaches: (i) in silico prediction, (ii) traditional in vitro functional assays across three signaling pathways with mutant-alone transfections, and (iii) modified in vitro assays with mutant and wild-type expression constructs co-transfected to model in vivo heterozygosity. We found that neither in silico analyses nor traditional in vitro assessments of mutants transfected alone could distinguish IGD variants from control variants. However, in vitro co-transfections revealed that 15/34 IGD variants caused loss-of-function (LoF), including 3 novel dominant-negatives, while only 4/25 control variants caused LoF. Surprisingly, 19 IGD-associated variants were benign or exhibited LoF that could be rescued by WT co-transfection. Overall, variants that were LoF in ≥ 2 signaling assays under co-transfection conditions were more likely to be disease-associated than benign or 'rescuable' variants. Our findings suggest that in vitro modeling of WT/Mutant interactions increases the resolution for identifying causal variants, uncovers novel dominant negative mutations, and provides new insights into the pathogenic mechanisms underlying heterozygous PROKR2 variants.
Assuntos
Nanismo Hipofisário/genética , Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Nanismo Hipofisário/metabolismo , Hormônio Liberador de Gonadotropina/deficiência , Células HEK293 , Humanos , Hipogonadismo/genética , Linhagem , Transdução de SinaisRESUMO
The acute effects of parathyroid hormone (PTH) on fibroblast growth factor 23 (FGF23) in vivo are not well understood. After a single subcutaneous PTH (1-34) injection (50 nmol/kg) in mice, FGF23 levels were assessed in plasma using assays that measure either intact alone (iFGF23) or intact/C-terminal FGF23 (cFGF23). Furthermore, FGF23 messenger RNA (mRNA) and protein levels were assessed in bone. In addition, we examined the effects of PTH treatment on FGF23 production in vitro using differentiated calvarial osteocyte-like cells. cFGF23 levels increased by three- to fivefold within 2 hours following PTH injection, which returned to baseline by 4 hours. In contrast, iFGF23 levels remained unchanged for the first 2 hours, yet declined to â¼60% by 6 hours and remained suppressed before returning to baseline after 24 hours. Using homozygous mice for an autosomal dominant hypophosphatemic rickets-FGF23 mutation or animals treated with a furin inhibitor, we showed that cFGF23 and iFGF23 levels increased equivalently after PTH injection. These findings are consistent with increased FGF23 production in bone, yet rapid cleavage of the secreted intact protein. Using primary osteocyte-like cell cultures, we showed that PTH increased FGF23 mRNA expression through cyclic adenosine monophosphate/protein kinase A, but not inositol triphosphate/protein kinase C signaling; PTH also increased furin protein levels. In conclusion, PTH injection rapidly increases FGF23 production in bone in vivo and in vitro. However, iFGF23 is rapidly degraded. At later time points through an unidentified mechanism, a sustained decrease in FGF23 production occurs.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Hormônio Paratireóideo/administração & dosagem , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/sangue , Domínios ProteicosRESUMO
Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Osteócitos/citologia , Osteócitos/metabolismo , Osteogênese/genética , Hormônio Paratireóideo/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ligante RANK/antagonistas & inibidores , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.
Assuntos
Conservadores da Densidade Óssea/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Modelos Moleculares , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Genes Reporter/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
GNAS, which encodes the stimulatory G protein (heterotrimeric guanine nucleotide-binding protein) α subunit (Gαs), also encodes a large variant of Gαs termed extra-large α subunit (XLαs), and alterations in XLαs abundance or activity are implicated in various human disorders. Although XLαs, like Gαs, stimulates generation of the second messenger cyclic adenosine monophosphate (cAMP), evidence suggests that XLαs and Gαs have opposing effects in vivo. We investigated the role of XLαs in mediating signaling by parathyroid hormone (PTH), which activates a G protein-coupled receptor (GPCR) that stimulates both Gαs and Gαq/11 in renal proximal tubules to maintain phosphate and vitamin D homeostasis. At postnatal day 2 (P2), XLαs knockout (XLKO) mice exhibited hyperphosphatemia, hypocalcemia, and increased serum concentrations of PTH and 1,25-dihydroxyvitamin D. The ability of PTH to reduce serum phosphate concentrations was impaired, and the abundance of the sodium phosphate cotransporter Npt2a in renal brush border membranes was reduced in XLKO mice, whereas PTH-induced cAMP excretion in the urine was modestly increased. Basal and PTH-stimulated production of inositol 1,4,5-trisphosphate (IP3), which is the second messenger produced by Gαq/11 signaling, was repressed in renal proximal tubules from XLKO mice. Crossing of XLKO mice with mice overexpressing XLαs specifically in renal proximal tubules rescued the phenotype of the XLKO mice. Overexpression of XLαs in HEK 293 cells enhanced IP3 generation in unstimulated cells and in cells stimulated with PTH or thrombin, which acts through a Gq/11-coupled receptor. Together, our findings suggest that XLαs enhances Gq/11 signaling to mediate the renal actions of PTH during early postnatal development.