Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice.

X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.

FGF23 promotes renal calcium reabsorption through the TRPV5 channel.

αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid-5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co-localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor-αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium-conserving hormone in the kidney.

FGF23 regulation of renal tubular solute transport.

PURPOSE OF REVIEW: Fibroblast growth factor-23 (FGF23) is a bone-derived hormone known to suppress phosphate reabsorption in the kidney. The purpose of this review was to highlight the recent advances in the area of FGF23-regulated solute transport in the kidney. RECENT FINDINGS: Recent evidence suggests that FGF23 suppresses phosphate reabsorption in renal proximal tubular epithelium by a Klotho-dependent, FGF receptor (FGFR)-1 and FGFR4-mediated signaling mechanism that may also involve Janus kinase 3. Moreover, it was recently established that FGF23 signaling in the distal renal tubule targets with-no-lysine kinase-4 (WNK4), a key molecule in the regulation of solute transport in the distal nephron. By targeting WNK4, FGF23 has been shown to increase the membrane abundance of the epithelial calcium channel TRPV5 and of the sodium-chloride cotransporter NCC, resulting in augmented renal calcium and sodium reabsorption. SUMMARY: Significant progress has been made in the further characterization of the signaling pathways involved in the FGF23-induced inhibition of phosphate transport in proximal tubular epithelium, and major new functions of FGF23 in solute transport have been discovered in distal renal tubules. The calcium- and sodium-conserving functions of FGF23 may have major implications for the pathophysiology of cardiovascular diseases. VIDEO ABSTRACT.

Single-dose GSTP1 prevents infarction-induced heart failure.

BACKGROUND: Heart failure (HF) is a common and often fatal complication of myocardial infarction (MI). Glutathione S-transferase P1-1 (GSTP1) has antiapoptotic and antiinflammatory effects and is a specific serum marker in HF patients. However, its role in HF treatment is unknown. METHODS AND RESULTS: GSTP1 effect was examined in a rat MI-induced HF model. Magnetic resonance imaging was used to examine cardiac function. GSTP1 and tumor necrosis factor α receptor-associated factor 2 (TRAF2) mRNA and protein expression were elevated in failing myocardium, although GSTP-1 binding activity to TRAF2 was not changed versus control. HF was associated with higher active JNK1 and p38 protein expression but reduced GSTP-1 binding activity to JNK1 and p38. Recombinant GSTP1 inhibited JNK1 and p38 and enhanced its own binding activity to TRAF2 and JNK1 in vitro. In the HF model, single-dose GSTP1 treatment reduced infarct area, apoptosis, and the expression of JNK1, p38, nuclear factor κB, and proinflammatory cytokines and improved thinning ratio, cardiac index and output, stroke volume, ejection fraction, regional wall motion, and survival compared with control. CONCLUSIONS: GSTP1 application early after MI results in long-term beneficial structural and functional effects that prevent progression to HF. GSTP1 could be a novel adjunct myocardial salvage approach in patients after MI.

Serum glutathione S-transferase P1 1 in prediction of cardiac function.

BACKGROUND: Glutathione S-transferase P1 1 (GSTP1) belongs to the multigene isozyme family involved in cellular response to oxidative stress and apoptosis. Our initial retrospective proteomic analysis suggested that GSTP1 is associated with heart failure (HF). Although pro-B-type natriuretic peptide (proBNP) serves currently as a surrogate diagnostic and prognostic parameter in HF patients, its specificity remains uncertain. We hypothesized that GSTP1 might be a useful serum marker in the monitoring of HF patients. METHODS AND RESULTS: Serum GSTP1 and proBNP were prospectively measured in 193 patients subdivided based on their ejection fraction (EF) either in equal-sized quintiles or predefined EF groups >52%, 43%-52%, 33%-42%, 23%-32% and ≤22%. At a cutoff of ≥231 ng/mL, GSTP1 identified HF patients with EF ≤22% with 81% sensitivity and 83% specificity, and at a cutoff of ≥655 pg/mL, proBNP identified the same patient group with 84% sensitivity and 22% specificity. GSTP1 at a ≥126 ng/mL cutoff identified EF ≤42% with 90% sensitivity and 95% specificity, or proBNP at a ≥396 pg/mL cutoff had 97% sensitivity and 20% specificity. In regression analyses, GSTP1, but not proBNP, discriminated between EF ≤42% and EF >42% in HF patients. CONCLUSIONS: These results suggest that GSTP1 is strongly associated with HF and could serve as a sensitive and specific marker to predict the ventricular function in HF patients.

Aldosterone Is Positively Associated With Circulating FGF23 Levels in Chronic Kidney Disease Across Four Species, and May Drive FGF23 Secretion Directly.

BACKGROUND: Chronic kidney disease (CKD) is accompanied by increases in circulating fibroblast growth factor 23 (FGF23) and aldosterone levels. Here, we tested the hypothesis that aldosterone may be one of the driving forces behind increased FGF23 secretion in CKD. METHODS: Using data from a prospective study in humans, a retrospective study in dogs and cats, and an experimental study in 5/6-nephrectomized mice, we analyzed the relationship between circulating FGF23 and serum aldosterone levels in CKD across four species. To assess the effects of acute inhibition of aldosterone signaling on circulating FGF23, we acutely treated mice with established CKD with the mineralocorticoid receptor blocker canrenone (50 mg/kg iv/sc), and measured intact FGF23 before and 24 h as well as 72 h after start of administration of the drug. RESULTS: We found a tight positive association between circulating intact FGF23 and serum aldosterone in human, canine, and feline CKD patients, as well as in experimental murine CKD (humans: r S = 0.57, p = 0.0368; dogs: r S = 0.66, p = 0.0019; cats: r S = 0.75, p = 0.0003; mice: r S = 0.49, p = 0.0004). Injection of canrenone in mice with established CKD did not lead to changes in FGF23 levels within 24 h, but reduced FGF23 in all mice at 72 h. CONCLUSION: Aldosterone may drive enhanced FGF23 secretion in CKD, possibly explaining the tight positive association between circulating intact FGF23 and aldosterone in human, canine, and feline CKD patients as well as in experimental CKD models.

The bone is the major source of high circulating intact fibroblast growth factor-23 in acute murine polymicrobial sepsis induced by cecum ligation puncture.

Fibroblast growth factor-23 (FGF23), a bone-produced hormone, plays a critical role in mineral homeostasis. Human diseases associated with excessive intact circulating FGF23 (iFGF23) result in hypophosphatemia and low vitamin D hormone in patients with normal kidney function. In addition, there is accumulating evidence linking FGF23 with inflammation. Based on these studies and the frequent observation of hypophosphatemia among septic patients, we sought to elucidate further the relationship between FGF23 and mineral homeostasis in a clinically relevant murine polymicrobial sepsis model. Medium-severity sepsis was induced by cecum ligation puncture (CLP) in adult CD-1 mice of both sexes. Healthy CD-1 mice (without CLP) were used as controls. Forty-eight hours post-CLP, spontaneous urine was collected, and serum, organs and bones were sampled at necropsy. Serum iFGF23 increased ~20-fold in CLP compared to control mice. FGF23 protein concentration was increased in the bones, but not in spleen or liver of CLP mice. Despite the ~20-fold iFGF23 increase, we did not observe any significant changes in mineral homeostasis or parathyroid hormone levels in the blood of CLP animals. Urinary excretion of phosphate, calcium, and sodium remained unchanged in male CLP mice, whereas female CLP mice exhibited lower urinary calcium excretion, relative to healthy controls. In line with renal FGF23 resistance, expression of phosphate-, calcium- and sodium-transporting proteins did not show consistent changes in the kidneys of male and female CLP mice. Renal expression of the co-receptor αKlotho was downregulated in female, but not in male CLP mice. In conclusion, our data demonstrate that the dramatic, sex-independent rise in serum iFGF23 post-CLP was mainly caused by an upregulation of FGF23 secretion in the bone. Surprisingly, the upsurge in circulating iFGF23 did not alter humoral mineral homeostasis in the acutely septic mice. Hence, the biological function of elevated FGF23 in sepsis remains unclear and warrants further studies.

Donor serum SMARCAL1 concentrations predict primary graft dysfunction in cardiac transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is a life-threatening complication in cardiac transplantation. A sensitive, specific, and easily measurable predictor in donors could facilitate PGD prevention. METHODS AND RESULTS: SMARCAL1 is a matrix-associated regulator of chromatin with helicase and ATPase activities, and its serum concentrations were significantly increased in a targeted protein array in donors whose grafts developed PGD. Therefore, this study analyzed SMARCAL1 serum concentrations by ELISA in 336 heart donors before and after aortic cross-clamping (ACC) and in recipients at 10, 30, and 60 minutes reperfusion. Demographic and hemodynamic parameters of donors and recipients as well as transplant procedure characteristics were documented. PGD (n=68) was defined as ventricular dilation and hypocontractility associated with systolic blood pressure <90 mm Hg, pulmonary capillary wedge pressure >20 mm Hg, and decreased mixed venous oxygen saturation necessitating mechanical circulatory support. SMARCAL1 serum protein concentration was significantly increased only before and after ACC in donors (P<0.0001) whose grafts developed PGD compared to those who did not. In receiver operating characteristic curve analysis, SMARCAL1 serum concentration at a cut-off level of > or =1.25 ng/mL before ACC in donors predicted PGD (P<0.0001, AUC=0.988, OR=17.050, 95% CI=5.200 to 55.901) with 96% sensitivity and 88% specificity. SMARCAL1 serum concentrations <1.25 ng/mL in donors before ACC resulted in 97% PGD-free outcome and SMARCAL1 concentrations > or =1.25 resulted in 83% PGD occurrence. CONCLUSIONS: Donor serum SMARCAL1 may serve as a specific, sensitive, and noninvasive predictive marker in the assessment of cardiac graft quality.

Fibrillar collagen inhibits cholesterol biosynthesis in human aortic smooth muscle cells.

OBJECTIVE: Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [(14)C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9+/-2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (-72.6+/-7.3%), mRNA (-58.7+/-6.4%), protein levels (-35.5+/-8.5%), and enzyme activity (-37.7+/-2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7+/-21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1+/-7.1%). The overexpression of the active form of SREBP1a rescued the downregulation of fibrillar collagen on HMG-CoA reductase levels. Blocking antibody to alpha2 integrin partially reversed the downregulation of HMG-CoA reductase mRNA expression. Finally, fibrillar collagen led to an intracellular accumulation of unprenylated Ras. CONCLUSIONS: Our study demonstrated that alpha2 beta 1 integrin interaction with fibrillar collagen affected the expression of HMG-CoA reductase, which led to the inhibition of cholesterol biosynthesis in human SMCs.

Augmented Fibroblast Growth Factor-23 Secretion in Bone Locally Contributes to Impaired Bone Mineralization in Chronic Kidney Disease in Mice.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic disorder of mineral and bone metabolism caused by CKD. Impaired bone mineralization together with increased bony secretion of fibroblast growth factor-23 (FGF23) are hallmarks of CKD-MBD. We recently showed that FGF23 suppresses the expression of tissue nonspecific alkaline phosphatase (TNAP) in bone cells by a Klotho-independent, FGF receptor-3-mediated signaling axis, leading to the accumulation of the mineralization inhibitor pyrophosphate. Therefore, we hypothesized that excessive FGF23 secretion may locally impair bone mineralization in CKD-MBD. To test this hypothesis, we induced CKD by 5/6 nephrectomy in 3-month-old wild-type (WT) mice and Fgf23-/-/VDRΔ/Δ (Fgf23/VDR) compound mutant mice maintained on a diet enriched with calcium, phosphate, and lactose. Eight weeks postsurgery, WT CKD mice were characterized by reduced bone mineral density at the axial and appendicular skeleton, hyperphosphatemia, secondary hyperparathyroidism, increased serum intact Fgf23, and impaired bone mineralization as evidenced by bone histomorphometry. Laser capture microdissection in bone cryosections showed that both osteoblasts and osteocytes contributed to the CKD-induced increase in Fgf23 mRNA abundance. In line with our hypothesis, osteoblastic and osteocytic activity of alkaline phosphatase was reduced, and bone pyrophosphate concentration was ~2.5-fold higher in CKD mice, relative to Sham controls. In Fgf23/VDR compound mice lacking Fgf23, 5/6-Nx induced secondary hyperparathyroidism and bone loss. However, 5/6-Nx failed to suppress TNAP activity, and bone pyrophosphate concentrations remained unchanged in Fgf23/VDR CKD mice. Collectively, our data suggest that elevated Fgf23 production in bone contributes to the mineralization defect in CKD-MBD by auto-/paracrine suppression of TNAP and subsequent accumulation of pyrophosphate in bone. Hence, our study has identified a novel mechanism involved in the pathogenesis of CKD-MBD.

Lack of vitamin D signalling per se does not aggravate cardiac functional impairment induced by myocardial infarction in mice.

Epidemiological studies have linked vitamin D deficiency to an increased incidence of myocardial infarction and support a role for vitamin D signalling in the pathophysiology of myocardial infarction. Vitamin D deficiency results in the development of secondary hyperparathyroidism, however, the role of secondary hyperparathyroidism in the pathophysiology of myocardial infarction is not known. Here, we aimed to explore further the secondary hyperparathyroidism independent role of vitamin D signalling in the pathophysiology of myocardial infarction by inducing experimental myocardial infarction in 3-month-old, male, wild-type mice and in mice lacking a functioning vitamin D receptor. In order to prevent secondary hyperparathyroidism in vitamin D receptor mutant mice, all mice were maintained on a rescue diet enriched with calcium, phosphorus, and lactose. Surprisingly, survival rate, cardiac function as measured by echocardiography and intra-cardiac catheterisation and cardiomyocyte size were indistinguishable between normocalcaemic vitamin D receptor mutant mice and wild-type controls, 2 and 8 weeks post-myocardial infarction. In addition, the myocardial infarction-induced inflammatory response was similar in vitamin D receptor mutants and wild-type mice, as evidenced by a comparable upregulation in cardiac interleukin-1-ß and tumor-necrosis-factor-α mRNA abundance and similar elevations in circulating interleukin-1-ß and tumor-necrosis-factor-α. Our data suggest that the lack of vitamin D signalling in normocalcaemic vitamin D receptor mutants has no major detrimental effect on cardiac function and outcome post-myocardial infarction. Our study may have important clinical implications because it suggests that the secondary hyperparathyroidism induced by vitamin D deficiency, rather than the lack of vitamin D signalling per se, may negatively impact cardiac function post-myocardial infarction.

Selective inhibition of receptor activator of NF-κB ligand (RANKL) in hematopoietic cells improves outcome after experimental myocardial infarction.

The RANK (receptor activator of nuclear factor κB)/RANKL (RANK ligand)/OPG (osteoprotegerin) axis is activated after myocardial infarction (MI), but its pathophysiological role is not well understood. Here, we investigated how global and cell compartment-selective inhibition of RANKL affects cardiac function and remodeling after MI in mice. Global RANKL inhibition was achieved by treatment of human RANKL knock-in (huRANKL-KI) mice with the monoclonal antibody AMG161. huRANKL-KI mice express a chimeric RANKL protein wherein part of the RANKL molecule is humanized. AMG161 inhibits human and chimeric but not murine RANKL. To dissect the pathophysiological role of RANKL derived from hematopoietic and mesenchymal cells, we selectively exchanged the hematopoietic cell compartment by lethal irradiation and across-genotype bone marrow transplantation between wild-type and huRANKL-KI mice, exploiting the specificity of AMG161. After permanent coronary artery ligation, mice were injected with AMG161 or an isotype control antibody over 4 weeks post-MI. MI increased RANKL expression mainly in cardiomyocytes and scar-infiltrating cells 4 weeks after MI. Only inhibition of RANKL derived from hematopoietic cellular sources, but not global or mesenchymal RANKL inhibition, improved post-infarct survival and cardiac function. Mechanistically, hematopoietic RANKL inhibition reduced expression of the pro-inflammatory cytokine IL-1ß in the cardiac cellular infiltrate. In conclusion, inhibition of RANKL derived from hematopoietic cellular sources is beneficial to maintain post-ischemic cardiac function by reduction of pro-inflammatory cytokine production. KEY MESSAGES: Experimental myocardial infarction (MI) augments cardiac RANKL expression in mice. RANKL expression is increased in cardiomyocytes and scar-infiltrating cells after MI. Global or mesenchymal cell RANKL inhibition has no influence on cardiac function after MI. Inhibition of RANKL derived from hematopoietic cells improves heart function post-MI. Hematopoietic RANKL inhibition reduces pro-inflammatory cytokines in scar-infiltrating cells.

FGF23-Klotho signaling axis in the kidney.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone protecting against the potentially deleterious effects of hyperphosphatemia by suppression of phosphate reabsorption and of active vitamin D hormone synthesis in the kidney. The kidney is one of the main target organs of FGF23 signaling. The purpose of this review is to highlight the recent advances in the area of FGF23-Klotho signaling in the kidney. During recent years, it has become clear that FGF23 acts independently on proximal and distal tubular epithelium. In proximal renal tubules, FGF23 suppresses phosphate reabsorption by a Klotho dependent activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and of serum/glucocorticoid-regulated kinase-1 (SGK1), leading to phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF)-1 and subsequent internalization and degradation of sodium-phosphate cotransporters. In distal renal tubules, FGF23 augments calcium and sodium reabsorption by increasing the apical membrane expression of the epithelial calcium channel TRPV5 and of the sodium-chloride cotransporter NCC through a Klotho dependent activation of with-no-lysine kinase-4 (WNK4). In proximal and distal renal tubules, FGF receptor-1 is probably the dominant FGF receptor mediating the effects of FGF23 by forming a complex with membrane-bound Klotho in the basolateral membrane. The newly described sodium- and calcium-conserving functions of FGF23 may have major implications for the pathophysiology of diseases characterized by chronically increased circulating FGF23 concentrations such as chronic kidney disease.

Response of human periodontal ligament stem cells to IFN-γ and TLR-agonists.

Periodontal ligament stem cells similarly to the mesenchymal stem cells of other tissues possess immunomodulatory properties, which are regulated by different cytokines, particularly by interferon-γ (IFN-γ). In contrast, less information is provided about the effect of toll-like receptors ligand on immunomodulatory properties of these cells. In the present study we investigated the response of human periodontal ligament stem cells (hPDLSCs) in response to simultaneous stimulation with IFN-γ and toll-like receptor (TLR) agonists. The resulting expression of indoleamine-2,3-dioxygenase-1 (IDO-1), interleukin (IL)-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was investigated. The expression of IDO-1 was upregulated by IFN-γ in both gene and protein levels. TLR2 agonists Pam3CSK4 induced gene expression of IDO-1, but had no effect on protein expression. IFN-γ induced IDO-1 protein expression was further enhanced by Pam3CSK4. TLR-4 agonist E. coli LPS has no significant effect on neither basal nor IFN-γ induced IDO-1 protein expression. The production of IL-6, IL-8, and MCP-1 was induced by TLR agonists. Neither basal nor TLR agonists induced production of these proteins was affected by IFN-γ. Our data shows potential interaction between IFN-γ and TLR2 responses in hPDLSCs, which might be involved in regulation of immune response in inflammatory diseases, and particularly periodontitis.

Genetic Ablation of Fgf23 or Klotho Does not Modulate Experimental Heart Hypertrophy Induced by Pressure Overload.

Left ventricular hypertrophy (LVH) ultimately leads to heart failure in conditions of increased cardiac pre- or afterload. The bone-derived phosphaturic and sodium-conserving hormone fibroblast growth factor-23 (FGF23) and its co-receptor Klotho have been implicated in the development of uremic LVH. Using transverse aortic constriction (TAC) in gene-targeted mouse models, we examine the role of Fgf23 and Klotho in cardiac hypertrophy and dysfunction induced by pressure overload. TAC profoundly increases serum intact Fgf23 due to increased cardiac and bony Fgf23 transcription and downregulation of Fgf23 cleavage. Aldosterone receptor blocker spironolactone normalizes serum intact Fgf23 levels after TAC by reducing bony Fgf23 transcription. Notably, genetic Fgf23 or Klotho deficiency does not influence TAC-induced hypertrophic remodelling, LV functional impairment, or LV fibrosis. Despite the profound, aldosterone-mediated increase in circulating intact Fgf23 after TAC, our data do not support an essential role of Fgf23 or Klotho in the pathophysiology of pressure overload-induced cardiac hypertrophy.

Klotho Lacks an FGF23-Independent Role in Mineral Homeostasis.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating vitamin D hormone production and renal handling of minerals by signaling through an FGF receptor/αKlotho (Klotho) receptor complex. Whether Klotho has FGF23-independent effects on mineral homeostasis is a controversial issue. Here, we aimed to shed more light on this controversy by comparing male and female triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) (Fgf23/Klotho/VDR) with double (Fgf23/VDR, Klotho/VDR, and Fgf23/Klotho) and single Fgf23, Klotho, and VDR mutants. As expected, 4-week-old Fgf23, Klotho, and Fgf23/Klotho knockout mice were hypercalcemic and hyperphosphatemic, whereas VDR, Fgf23/VDR, and Klotho/VDR mice on rescue diet were normocalcemic and normophosphatemic. Serum levels of calcium, phosphate, and sodium did not differ between 4-week-old triple Fgf23/Klotho/VDR and double Fgf23/VDR or Klotho/VDR knockout mice. Notably, 3-month-old Fgf23/Klotho/VDR triple knockout mice were indistinguishable from double Fgf23/VDR and Klotho/VDR compound mutants in terms of serum calcium, serum phosphate, serum sodium, and serum PTH, as well as urinary calcium and sodium excretion. Protein expression analysis revealed increased membrane abundance of sodium-phosphate co-transporter 2a (NaPi-2a), and decreased expression of sodium-chloride co-transporter (NCC) and transient receptor potential cation channel subfamily V member 5 (TRPV5) in Fgf23/Klotho/VDR, Fgf23/VDR, and Klotho/VDR mice, relative to wild-type and VDR mice, but no differences between triple and double knockouts. Further, ex vivo treatment of live kidney slices isolated from wild-type and Klotho/VDR mice with soluble Klotho did not induce changes in intracellular phosphate, calcium or sodium accumulation assessed by two-photon microscopy. In conclusion, our data suggest that the main physiological function of Klotho for mineral homeostasis in vivo is its role as co-receptor mediating Fgf23 action. © 2017 American Society for Bone and Mineral Research.

Acute Parathyroid Hormone Injection Increases C-Terminal but Not Intact Fibroblast Growth Factor 23 Levels.

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 ∼60% 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.

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Fgf23 and parathyroid hormone signaling interact in kidney and bone.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone, suppressing renal phosphate reabsorption and vitamin D hormone synthesis in proximal tubules, and stimulating calcium reabsorption in distal tubules of the kidney. Here, we analyzed the long term sequelae of deficient Fgf23 signaling on bone and mineral metabolism in 9-month-old mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor (VDR). To prevent hypocalcemia in VDR deficient mice, all mice were kept on a rescue diet enriched with calcium, phosphate, and lactose. VDR mutants were normocalcemic and normophosphatemic, and had normal tibial bone mineral density. Relative to VDR mutants, Fgf23/VDR and Klotho/VDR compound mutants were characterized by hypocalcemia, hyperphosphatemia, and very high serum parathyroid hormone (PTH). Despite ∼10-fold higher serum PTH levels in compound mutants, urinary excretion of phosphate and calcium as well as osteoclast numbers in bone remained unchanged relative to VDR mutants. The increase in plasma cAMP after hPTH(1-34) injection was similar in all genotypes. However, a 5-day infusion of hPTH(1-34) via osmotic minipumps resulted in reduced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in bone and kidney of Fgf23/VDR and Klotho/VDR compound mutants, relative to VDR and WT controls. Similarly, the PTH-mediated ERK1/2 phosphorylation was reduced in primary osteoblasts isolated from Fgf23 and Klotho deficient mice, but was restored by concomitant treatment with recombinant FGF23. Collectively, our data indicate that the phosphaturic, calcium-conserving, and bone resorption-stimulating actions of PTH are blunted by Fgf23 or Klotho deficiency. Hence, FGF23 may be an important modulator of PTH signaling in bone and kidney.