Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Structural asymmetry in FGF23 signaling.

Chen et al. have derived cryogenic electron microscopy (cryo-EM) structures of signaling complexes of the endocrine hormone fibroblast growth factor 23 (FGF23) with fibroblast growth factor receptor (FGFR), α-Klotho, and heparin sulfate. These structures are asymmetric, leading to questions concerning in vivo function, and will facilitate structure-based drug design to modulate FGF23 signaling.

Identification of Small-Molecule Inhibitors of Fibroblast Growth Factor 23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho.

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, the FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, burosumab. Small-molecule drugs that disrupt protein/protein interactions necessary for the ternary complex formation offer an alternative to disrupting FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein/protein interaction inhibitors since it was computationally predicted to have a large fraction of hot spots and two druggable residues on α-Klotho. We further identified Tyr433 on the KL1 domain of α-Klotho as a promising hot spot and α-Klotho as an appropriate drug-binding target at this interface. Subsequently, we performed in silico docking of ∼5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 24 and 20 compounds were in the final list based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, respectively. Five compounds were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduced activities significantly. Both these compounds were predicted to have favorable binding affinities to α-Klotho but not have a large number of contacts with the hot spot Tyr433. ZINC12409120 was found experimentally to disrupt FGF23:α-Klotho interaction to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 µM. Molecular dynamics (MD) simulations of the ZINC12409120:α-Klotho complex starting from in silico docking poses reveal that the ligand exhibits contacts with residues on the KL1 domain, the KL1-KL2 linker, and the KL2 domain of α-Klotho simultaneously, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23:α-Klotho interaction. ZINC12409120 is a candidate for lead optimization.

Novel Treatments from Inhibition of the Intestinal Sodium-Hydrogen Exchanger 3.

Plasma membrane sodium-hydrogen exchangers (NHE) transport Na+ into cells in exchange for H+. While there are nine isoforms of NHE in humans, this review focuses on the NHE3 isoform, which is abundantly expressed in the gastrointestinal tract, where it plays a key role in acid-base balance and water homeostasis. NHE3 inhibition in the small intestine results in luminal sodium and water retention, leading to a general decrease in paracellular water flux and diffusional driving force, reduced intestinal sodium absorption, and increased stool sodium excretion. The resulting softer and more frequent stools are the rationale for the development of tenapanor as a novel, first-in-class NHE3 inhibitor to treat irritable bowel syndrome with constipation. NHE3 also has additional therapeutic implications in nephrology. Inhibition of intestinal NHE3 also lowers blood pressure by reducing intestinal sodium absorption. Perhaps, the most novel effect is its ability to decrease intestinal phosphate absorption by inhibiting the paracellular phosphate absorption pathway. Therefore, selective pharmacological inhibition of NHE3 could be a potential therapeutic strategy to treat not only heart failure and hypertension but also hyperphosphatemia. This review presents an overview of the molecular and physiological functions of NHE3 and discusses how these functions translate to potential clinical applications in nephrology.

Osteoporosis: Mechanism, Molecular Target and Current Status on Drug Development.

CDATA[Osteoporosis is a pathological loss of bone mass due to an imbalance in bone remodeling where osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation resulting in skeletal fragility and fractures. Anti-resorptive agents, such as bisphosphonates and SERMs, and anabolic drugs that stimulate bone formation, including PTH analogues and sclerostin inhibitors, are current treatments for osteoporosis. Despite their efficacy, severe side effects and loss of potency may limit the long term usage of a single drug. Sequential and combinational use of current drugs, such as switching from an anabolic to an anti-resorptive agent, may provide an alternative approach. Moreover, there are novel drugs being developed against emerging new targets such as Cathepsin K and 17ß-HSD2 that may have less side effects. This review will summarize the molecular mechanisms of osteoporosis, current drugs for osteoporosis treatment, and new drug development strategies.

Humanized GPRC6AKGKY is a gain-of-function polymorphism in mice.

GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.

FGF23 induced left ventricular hypertrophy mediated by FGFR4 signaling in the myocardium is attenuated by soluble Klotho in mice.

There is controversy regarding whether excess FGF23 causes left ventricular hypertrophy (LVH) directly through activation of fibroblast growth factor receptor 4 (FGFR4) in cardiomyocytes or indirectly through reductions in soluble Klotho (sK). We investigated the respective roles of myocardial FGFR4 and sKL in mediating FGF23-induced LVH using mouse genetic and pharmacological approaches. To investigate a direct role of myocardial FGFR4 in mediating the cardiotoxic effects of excess circulating FGF23, we administered rFGF23 to mice with cardiac-specific loss of FGFR4 (FGFR4 heart-cKO). We tested a model of sKL deficiency, hypertension and LVH created by the conditional deletion of FGFR1 in the renal distal tubule (FGFR1DT cKO mice). The cardioprotective effects of sKL in both mouse models was assessed by the systemic administration of recombinant sKL. We confirmed that FGF23 treatment activates PLCγ in the heart and induces LVH in the absence of membrane α-Klotho. Conditional deletion of FGFR4 in the myocardium prevented rFGF23-induced LVH in mice, establishing direct cardiotoxicity of FGF23 through activation of FGFR4. Recombinant sKL administration prevented LVH, but not HTN, in FGFR1DT cKO mice, consistent with direct cardioprotective effects. Co-administration of recombinant sKL with FGF23 in culture inhibited rFGF23-induced p-PLCγ signaling. Thus, FGF23 ability to include LVH represents a balance between FGF23 direct cardiac activation of FGFR4 and the modulating effects of circulating sKL to alter FGF23-dependent myocardial signaling pathways.

Molecular Control of Phosphorus Homeostasis and Precision Treatment of Hypophosphatemic Disorders.

PURPOSE OF REVIEW: Serum phosphorus is maintained in a narrow range by balancing dietary phosphate absorption, influx and efflux of phosphorus from bone and intracellular stores, and renal reabsorption of filtered phosphate. Acute hypophosphatemia, typically caused by transient increases in cellular uptake, can lead to severe complications such as cardiopulmonary dysfunction and rhabdomyolysis that can warrant parenteral phosphate repletion. Chronic hypophosphatemia, however, generally represents true phosphate deficiency and may result in long-term metabolic and skeletal complications, particularly in children due to the critical importance of phosphorus to skeletal mineralization and longitudinal growth. RECENT FINDINGS: In addition to the well characterized roles of vitamin D and parathyroid hormone (PTH), a new bone-kidney axis has been discovered that regulates phosphate homeostasis through the bone-derived hormone Fibroblast Growth Factor 23 (FGF23) and its phosphaturic actions that are mediated by activation of fibroblast growth factor receptors (FGFRs) complexed with α-Klotho in renal tubules. Chronic hypophosphatemia can now be classified as FGF23 dependent or independent. SUMMARY: In cases of FGF23 dependent hypophosphatemia, traditional non-specific treatments with elemental phosphorus and 1,25(OH)2 vitamin D (calcitriol) can now be replaced with a targeted approach by using an FGF-23 blocking antibody (Burosumab).

Human GPRC6A Mediates Testosterone-Induced Mitogen-Activated Protein Kinases and mTORC1 Signaling in Prostate Cancer Cells.

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6AICL3_KGKY) that replaces the ancestral KGRKLP sequence (GPRC6AICL3_RKLP) present in all other species. In vitro assays purport that human GPRC6AICL3_KGKY is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6AICL3_KGKY in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY), and humanized mouse (mGPRC6AICL3_KGKY) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6AICL3_KGRKLP acts as a classic G protein-coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6AICL3_KGKY and humanized mouse mGPRC6AICL3_KGKY are retained intracellularly in ligand naive cells, yet exhibit ß-arrestin-dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal-regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6AICL3_KGKY is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6AICL3_KGKY In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A-deficient cells, the effect of testosterone to activate downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.

FGF-23 Deficiency Impairs Hippocampal-Dependent Cognitive Function.

Fibroblast growth factor receptor (FGFR) and α-Klotho transduce FGF-23 signaling in renal tubules to maintain systemic phosphate/vitamin D homeostasis. Mice deficient for either the ligand, FGF-23, or the co-receptor, Klotho, are phenocopies with both showing rapid and premature development of multiple aging-like abnormalities. Such similarity in phenotype, suggests that FGF-23 and Klotho have co-dependent systemic functions. Recent reports revealed inverse central nervous system (CNS) effects of Klotho deficiency or Klotho overexpression on hippocampal synaptic, neurogenic, and cognitive functions. However, it is unknown whether FGF-23 deficiency effects function of the hippocampus. We report that, similar to Klotho-deficient mice, FGF-23-deficient mice develop dose-dependent, hippocampal-dependent cognitive impairment. However, FGF-23-deficient brains had no gross structural or developmental defects, no change in hippocampal synaptic plasticity, and only minor impairment to postnatal hippocampal neurogenesis. Together, these data provide evidence that FGF-23 deficiency impairs hippocampal-dependent cognition but otherwise results in a brain phenotype that is distinct from the KL-deficient mouse.

Fibroblast growth factor 23 and α-Klotho co-dependent and independent functions.

PURPOSE OF REVIEW: The current review examines what is known about the FGF-23/α-Klotho co-dependent and independent pathophysiological effects, and whether FGF-23 and/or α-Klotho are potential therapeutic targets. RECENT FINDINGS: FGF-23 is a hormone derived mainly from bone, and α-Klotho is a transmembrane protein. Together they form a trimeric signaling complex with FGFRs in target tissues to mediate the physiological functions of FGF-23. Local and systemic factors control FGF-23 release from osteoblast/osteocytes in bone, and circulating FGF-23 activates FGFR/α-Klotho complexes in kidney proximal and distal renal tubules to regulate renal phosphate excretion, 1,25 (OH)2D metabolism, sodium and calcium reabsorption, and ACE2 and α-Klotho expression. The resulting bone-renal-cardiac-immune networks provide a new understanding of bone and mineral homeostasis, as well as identify other biological effects FGF-23. Direct FGF-23 activation of FGFRs in the absence of α-Klotho is proposed to mediate cardiotoxic and adverse innate immune effects of excess FGF-23, particularly in chronic kidney disease, but this FGF-23, α-Klotho-independent signaling is controversial. In addition, circulating soluble Klotho (sKl) released from the distal tubule by ectodomain shedding is proposed to have beneficial health effects independent of FGF-23. SUMMARY: Separation of FGF-23 and α-Klotho independent functions has been difficult in mammalian systems and understanding FGF-23/α-Klotho co-dependent and independent effects are incomplete. Antagonism of FGF-23 is important in treatment of hypophosphatemic disorders caused by excess FGF-23, but its role in chronic kidney disease is uncertain. Administration of recombinant sKl is an unproven therapeutic strategy that theoretically could improve the healt span and lifespan of patients with α-Klotho deficiency.

Validation of a Novel Modified Aptamer-Based Array Proteomic Platform in Patients with End-Stage Renal Disease.

End stage renal disease (ESRD) is characterized by complex metabolic abnormalities, yet the clinical relevance of specific biomarkers remains unclear. The development of multiplex diagnostic platforms is creating opportunities to develop novel diagnostic and therapeutic approaches. SOMAscan is an innovative multiplex proteomic platform which can measure >1300 proteins. In the present study, we performed SOMAscan analysis of plasma samples and validated the measurements by comparison with selected biomarkers. We compared concentrations of SOMAscan-measured prostate specific antigen (PSA) between males and females, and validated SOMAscan concentrations of fibroblast growth factor 23 (FGF23), FGF receptor 1 (FGFR1), and FGFR4 using Enzyme-Linked immunosorbent assay (ELISA). The median (25th and 75th percentile) SOMAscan PSA level in males and females was 4304.7 (1815.4 to 7259.5) and 547.8 (521.8 to 993.4) relative fluorescence units (p = 0.002), respectively, suggesting biological plausibility. Pearson correlation between SOMAscan and ELISA was high for FGF23 (R = 0.95, p < 0.001) and FGFR4 (R = 0.69, p < 0.001), indicating significant positive correlation, while a weak correlation was found for FGFR1 (R = 0.13, p = 0.16). In conclusion, there is a good to near-perfect correlation between SOMAscan and standard immunoassays for FGF23 and FGFR4, but not for FGFR1. This technology may be useful to simultaneously measure a large number of plasma proteins in ESRD, and identify clinically important prognostic markers to predict outcomes.

Cardiovascular Interactions between Fibroblast Growth Factor-23 and Angiotensin II.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5-1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

Changes With Lanthanum Carbonate, Calcium Acetate, and Phosphorus Restriction in CKD: A Randomized Controlled Trial.

INTRODUCTION: Abnormal phosphorus homeostasis develops early in chronic kidney disease (CKD). It is unclear if its correction results in improved clinical outcomes in non-dialysis dependent CKD. METHODS: We conducted a randomized controlled, parallel design clinical trial in 120 patients with estimated glomerular filtration rate 15 to 59 ml/min per 1.73 m2 and abnormal phosphorus homeostasis (serum phosphorus >4.6 mg/dl, parathyroid hormone [PTH] >70 pg/ml or tubular reabsorption of phosphorus [TRP] <80%). Patients were randomized to open-label lanthanum carbonate versus calcium acetate versus dietary intervention over 1 year. The co-primary outcomes were month 12 (vs. baseline) biochemical (serum phosphorus, TRP, PTH, calcium, bone-specific alkaline phosphatase [bALP], and fibroblast growth factor 23 [FGF23]) and vascular parameters (coronary artery calcium score, pulse wave velocity, and endothelial dysfunction) in all patients. Secondary outcomes were between-treatment differences in change for each parameter between month 12 and baseline. All analyses were intention to treat. RESULTS: Baseline characteristics were similar in the 3 groups. A total of 107 patients (89%) completed 12 months of follow-up. Differences were not significant at month 12 (vs. baseline) for any of the outcomes except bALP (median [25th, 75th] percentile at month 12 versus baseline: 13.8 [10.6, 17.6] vs. 15.8 [12.1, 21.1], P < .001) and FGF23 (132 [99, 216] vs. 133 [86, 189], P = .002). Changes for all outcomes were similar in the 3 arms except for PTH, which was suppressed more effectively by calcium acetate (P < .001). CONCLUSION: A 1-year intervention to limit phosphorus absorption using dietary restriction or 2 different phosphorus binders resulted in decreased bALP suggesting improved bone turnover, but no other significant changes in biochemical or vascular parameters in patients with CKD stage 3/4. (ClinicalTrials.gov: NCT01357317).

Role of Fibroblast Growth Factor-23 in Innate Immune Responses.

Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone that activates FGFR/α-Klotho binary complexes in the kidney renal tubules to regulate phosphate reabsorption and vitamin D metabolism. The objective of this review is to discuss the emerging data that show that FGF-23 has functions beyond regulation of mineral metabolism, including roles in innate immune and hemodynamic responses. Excess FGF-23 is associated with inflammation and adverse infectious outcomes, as well as increased morbidity and mortality, particularly in patients with chronic kidney disease. Enhancer elements in the FGF-23 promoter have been identified that mediate the effects of inflammatory cytokines to stimulate FGF-23 gene transcription in bone. In addition, inflammation induces ectopic expression of FGF-23 and α-Klotho in macrophages that do not normally express FGF-23 or its binary receptor complexes. These observations suggest that FGF-23 may play an important role in regulating innate immunity through multiple potential mechanisms. Circulating FGF-23 acts as a counter-regulatory hormone to suppress 1,25D production in the proximal tubule of the kidney. Since vitamin D deficiency may predispose infectious and cardiovascular diseases, FGF-23 effects on innate immune responses may be due to suppression of 1,25D production. Alternatively, systemic and locally produced FGF-23 may modulate immune functions through direct interactions with myeloid cells, including macrophages and polymorphonuclear leukocytes to impair immune cell functions. Short-acting small molecules that reversibly inhibit FGF-23 offer the potential to block pro-inflammatory and cardiotoxic effects of FGF-23 with less side effects compared with FGF-23 blocking antibodies that have the potential to cause hyperphosphatemia and soft tissue calcifications in animal models. In conclusion, there are several mechanisms by which FGF-23 impacts the innate immune system and further investigation is critical for the development of therapies to treat diseases associated with elevated FGF-23.

Computationally identified novel agonists for GPRC6A.

New insights into G protein coupled receptor regulation of glucose metabolism by ß-cells, skeletal muscle and liver hepatocytes identify GPRC6A as a potential therapeutic target for treating type 2 diabetes mellitus (T2D). Activating GPRC6A with a small molecule drug represents a potential paradigm-shifting opportunity to make significant strides in regulating glucose homeostasis by simultaneously correcting multiple metabolic derangements that underlie T2D, including abnormalities in ß-cell proliferation and insulin secretion and peripheral insulin resistance. Using a computational, structure-based high-throughput screening approach, we identified novel tri-phenyl compounds predicted to bind to the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A. Experimental testing found that these compounds dose-dependently stimulated GPRC6A signaling in a heterologous cell expression system. Additional chemical modifications and functional analysis identified one tri-phenyl lead compound, DJ-V-159 that demonstrated the greatest potency in stimulating insulin secretion in ß-cells and lowering serum glucose in wild-type mice. Collectively, these studies show that GPRC6A is a "druggable" target for developing chemical probes to treat T2DM.

Cardiovascular Effects of Renal Distal Tubule Deletion of the FGF Receptor 1 Gene.

The bone-derived hormone fibroblast growth factor-23 (FGF-23) activates complexes composed of FGF receptors (FGFRs), including FGFR1, and α-Klotho in the kidney distal tubule (DT), leading to increased sodium retention and hypertension. However, the role of FGFR1 in regulating renal processes linked to hypertension is unclear. Here, we investigated the effects of selective FGFR1 loss in the DT. Conditional knockout (cKO) of FGFR1 in the DT (FGFR1DT-cKO mice) resulted in left ventricular hypertrophy (LVH) and decreased kidney expression of α-Klotho in association with enhanced BP, decreased expression of angiotensin converting enzyme 2, and increased expression of the Na+-K+-2Cl- cotransporter. Notably, recombinant FGF-23 administration similarly decreased the kidney expression of α-Klotho and induced LVH in mice. Pharmacologic activation of FGFR1 with a monoclonal anti-FGFR1 antibody (R1MAb1) normalized BP and significantly attenuated LVH in the Hyp mouse model of excess FGF-23, but did not induce a response in FGFR1DT-cKO mice. The hearts of FGFR1DT-cKO mice showed increased expression of the transient receptor potential cation channel, subfamily C, member 6 (TRPC6), consistent with cardiac effects of soluble Klotho deficiency. Moreover, administration of recombinant soluble Klotho lowered BP in the Hyp mice. Thus, FGFR1 in the DT regulates systemic hemodynamic responses opposite to those predicted by the actions of FGF-23. These cardiovascular effects appear to be mediated by paracrine FGF control of kidney FGFR1 and subsequent regulation of soluble Klotho and TRPC6. FGFR1 in the kidney may provide a new molecular target for treating hypertension.

Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis.

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.

CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model.

BACKGROUND: GPRC6A is implicated in the pathogenesis of prostate cancer, but its role remains uncertain because of a purported tolerant gene variant created by substitution of a K..Y polymorphism in the 3rd intracellular loop (IL) that evolved in the majority of humans and replaces the ancestral RKLP present in 40% of humans of African descent and all other species. METHODS: We determined whether the K..Y polymorphism is present in human-derived prostate cancer cell lines by sequencing the region of the 3rd IL and assessed the cellular localization of a "humanized" mouse GPRC6A containing the K..Y sequence by immunofluorescence. We assessed functions of GPRC6A in PC-3 cells expressing endogenous GPRC6A and in GPRC6A-deficient PC-3 cells created using CRISPR/Cas9 technology. The effect of GPRC6A on basal and ligand stimulated cell proliferation and migration was evaluated in vitro in wild-type and PC-3-deficient cell lines. The effect of editing GPRC6A on prostate cancer growth and progression in vivo was assessed in a Xenograft mouse model implanted with wild-type and PC-3 deficient cells and treated with the GPRC6A ligand osteocalcin. RESULTS: We found that all of the human prostate cancer cell lines tested endogenously express the "K..Y" polymorphism in the 3rd IL. Comparison of mouse wild-type GPRC6A with a "humanized" mouse GPRC6A construct created by replacing the "RKLP" with the "K..Y" sequence, found that both receptors were predominantly expressed on the cell surface. The transfected "humanized" GPRC6A receptor, however, preferentially activated mTOR compared to ERK signaling in HEK-293 cells. In contrast, in PC-3 cells expressing the endogenous GPRC6A with the "K..Y" polymorphism, the ligand osteocalcin stimulated ERK, AKT and mTOR phosphorylation, promoted cell proliferation and migration, and upregulated genes regulating testosterone biosynthesis. Targeting GPRC6A in PC-3 cells by CRISPR/Cas9 significantly blocked these responses in vitro. In addition, GPRC6A deficient PC-3 xenografts exhibited significantly less growth and were resistant to osteocalcin-induced prostate cancer progression compared to control PC-3 cells expressing GPRC6A. CONCLUSIONS: Human GPRC6A is a functional osteocalcin and testosterone sensing receptor that promotes prostate cancer progression. GPRC6A may contribute to racial disparities in prostate cancer, and is a potential therapeutic target to develop antagonists to treat prostate cancer.