Claudin 18 is a novel negative regulator of bone resorption and osteoclast differentiation

Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18(-/-) mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18(-/-) mice without changes in bone formation. Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation but not macrophage colony-stimulating factor (MCSF)-induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation.     

DLK1 is a novel regulator of bone mass that mediates estrogen deficiency–induced bone loss in mice

Delta‐like 1/fetal antigen 1 (DLK1/FA‐1) is a transmembrane protein belonging to the Notch/Delta family that acts as a membrane‐associated or a soluble protein to regulate regeneration of a number of adult tissues. Here we examined the role of DLK1/FA‐1 in bone biology using osteoblast‐specific Dlk1‐overexpressing mice (Col1‐Dlk1). Col1‐Dlk1 mice displayed growth retardation and significantly reduced total body weight and bone mineral density (BMD). Micro–computed tomographis (µCT) scanning revealed a reduced trabecular and cortical bone volume fraction. Tissue‐level histomorphometric analysis demonstrated decreased bone‐formation rate and enhanced bone resorption in Col1‐Dlk1 mice compared with wild‐type mice. At a cellular level, Dlk1 markedly reduced the total number of bone marrow (BM)–derived colony‐forming units fibroblasts (CFU‐Fs), as well as their osteogenic capacity. In a number of in vitro culture systems, Dlk1 stimulated osteoclastogenesis indirectly through osteoblast‐dependent increased production of proinflammatory bone‐resorbing cytokines (eg, Il7, Tnfa, and Ccl3). We found that ovariectomy (ovx)–induced bone loss was associated with increased production of Dlk1 in the bone marrow by activated T cells. Interestingly, Dlk1^?/? mice were significantly protected from ovx‐induced bone loss compared with wild‐type mice. Thus we identified Dlk1 as a novel regulator of bone mass that functions to inhibit bone formation and to stimulate bone resorption. Increasing DLK1 production by T cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss. © 2011 American Society for Bone and Mineral Research.     

MEPE is a novel regulator of growth plate cartilage mineralization

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and μCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide.     

Melanin concentrating hormone is a novel regulator of islet function and growth

Melanin concentrating hormone (MCH) is a hypothalamic neuropeptide known to play a critical role in energy balance. We have previously reported that overexpression of MCH is associated with mild obesity. In addition, mice have substantial hyperinsulinemia and islet hyperplasia that is out of proportion with their degree of obesity. In this study, we further explored the role of MCH in the endocrine pancreas. Both MCH and MCHR1 are expressed in mouse and human islets and in clonal beta-cell lines as assessed using quantitative real-time PCR and immunohistochemistry. Mice lacking MCH (MCH-KO) on either a C57Bl/6 or 129Sv genetic background showed a significant reduction in beta-cell mass and complemented our earlier observation of increased beta-cell mass in MCH-overexpressing mice. Furthermore, the compensatory islet hyperplasia secondary to a high-fat diet, which was evident in wild-type controls, was attenuated in MCH-KO. Interestingly, MCH enhanced insulin secretion in human and mouse islets and rodent beta-cell lines in a dose-dependent manner. Real-time PCR analyses of islet RNA derived from MCH-KO revealed altered expression of islet-enriched genes such as glucagon, forkhead homeobox A2, hepatocyte nuclear factor (HNF)4alpha, and HNF1alpha. Together, these data provide novel evidence for an autocrine role for MCH in the regulation of beta-cell mass dynamics and in islet secretory function and suggest that MCH is part of a hypothalamic-islet (pancreatic) axis.     

Teneurin‐4, a transmembrane protein,is a novel regulator that suppresses chondrogenic differentiation

Teneurin‐4 (Ten‐4), a transmembrane protein, is expressed in the nervous systems and the mesenchymal tissues, including the cartilage. However, the Ten‐4 function in cartilage development remains unknown. Here, we showed that Ten‐4 is a novel regulator of chondrogenesis. In situ hybridization analysis revealed that Ten‐4 was highly expressed in the mesenchymal condensation area of the mouse femur at embryonic day (E) 13.5, while its expression was decreased in the growth plate of the femur at E18.5. Using the cartilage‐like pellet culture of human synovial mesenchymal cells, Ten‐4 expression was induced and peaked 7 days after induction of differentiation, while a production of type II and X collagens was increased after Day 14. In the cartilage‐like pellet, Ten‐4 was highly expressed in the less differentiated region. In the chondrogenic cell line ATDC5, knockdown of Ten‐4 expression significantly increased the alcian blue staining and expression levels of aggrecan and type II and X collagens. Further, an elevated expression of Sox6, Sox9, and Runx2 and an attenuation of the ERK activation were observed in the Ten‐4‐knockdown ATDC5 cells. These results suggested that Ten‐4 suppresses chondrogenic differentiation and regulates the expression and activation of the key molecules for chondrogenesis. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:915–922, 2014.     

The duration of exercise as a regulator of bone turnover

The relationship between duration of exercise and serum remodeling markers of bone turnover was evaluated by osteocalcin (OC), carboxy-terminal propeptide of type I collagen (PICP), total and bone-specific alkaline phosphatase (ALP) and carboxyterminal cross-linked telopeptide of type I collagen (ICTP) in 24 male premier league soccer players exercising 12 hours/week (range 8–18), 19 third league players exercising 8 hours/week (range 3–18) and 20 sixth league players exercising 6 hours/week (range 2–10). Twenty-seven volunteers served as controls. Forty-six former male soccer players (mean age 38 years, range 19–47), mean 15 years older than the current players, were compared with 41 matched controls. Data is presented as mean ± SEM. Active male players had 18 ± 4% higher OC, 37 ± 9% higher bone ALP and 36 ± 7% higher ICTP than controls (all P < 0.01). There were no differences in remodeling markers within the three groups of active players but each group had higher OC and ICTP than controls (both P < 0.05). Former players had no difference in bone remodeling markers compared to matched controls, but 39 ± 4% lower OC and 69 ± 8% lower ICTP than active players (both P < 0.001). Duration of activity was correlated with bone ALP and ICTP (both r = 0.3, P < 0.05) in individuals exercising 6 hours/week or less. No correlation was found in those exercising above this level. It seems as if the bone turnover, evaluated by serum bone remodeling markers, adapts to the current activity needed to maintain bone strength, and a duration of exercise above that level seems to confer no additional benefits.     

The duration of exercise as a regulator of bone mass

Exercise is associated with increased peak bone mineral density (BMD). To determine the relationship between the duration of exercise and BMD, we measured BMD of the axial and appendicular skeleton by dual-energy X-ray absorptiometry (DXA), and speed of sound (SOS), broadband attenuation (BUA), and stiffness index by quantitative ultrasound (QUS) of the calcaneus, in 67 active male national soccer players (mean age 23 years, range 17-35), which included 23 premier-league players exercising 12 h/week (range 8-18), 23 third-league players exercising 8 h/week (range 3-18), and 21 sixth-league players exercising 6 h/week (range 2-10). Results were compared with 24 sedentary age- and gender-matched controls and presented as mean +/- SEM. BMD was higher in all weight-bearing regions for the whole group relative to controls (BMD: total body 6.8 +/- 0.7%, leg 9.6 +/- 0.8%, lumbar spine 13.2 +/- 1.2%, femoral neck 12.7 +/- 1.2% [all p < 0.001]; calcaneus SOS 4.2 +/- 0.3%, BUA 8.7 +/- 1.5%, and stiffness index 24.2 +/- 2.0% [all p < 0.01]). No differences were found in head or arm BMD. There were no differences in BMD or QUS measurements when comparing soccer players exercising for different activity durations. Duration of activity correlated with BMD weight-loaded regions and with QUS, provided it was less <6 h/week (p < 0.01 respectively), but not when exercising more frequently. Femoral neck BMD increased by 3.3% across every hour increase in activity in those with 0-6 h of exercise/week and by 0.7% in those exercising more than this (p < 0.01). We conclude that, in national-league soccer, the BMD needed to attain a bone strength commensurate with that of duration of activity is achieved by 6 h of exercise per week. Beyond this, additional exercise confers no higher BMD. The skeleton adapts to the prevalent level of exercise intensity required and no further.     

Renalase, a novel regulator of blood pressure, is predicted by kidney function in renal transplant recipients

Background

Renalase is an enzyme that catabolizes catecholamines such as adrenaline and noradrenaline in the circulation. The human kidney releases this protein into the bloodstream to regulate blood pressure. In kidney transplant recipients, the prevalence of hypertension is 60%-80%.

Objective

The aim of our study was to assess possible correlations between renalase, blood pressure, and kidney function among 89 prevalent kidney allograft recipients. To obtain normal ranges, we also studied renalase levels in 27 healthy volunteers.

Methods

Complete blood counts, urea, serum lipids, fasting glucose, and creatinine were measured by standard laboratory methods in the hospital central laboratory. Renalase was assessed with the use of a commercially available kit.

Results

In kidney transplant recipients renalase was significantly higher than in healthy volunteers (P < .001). In kidney transplant recipients, renalase correlated with age (r = 0.29; P < .05), time after transplantation (r = 0.34; P < .01), systolic blood pressure (r = 0.28; P < .05), diastolic blood pressure (r = 0.27; P < .05), serum creatinine (r = 0.49; P < .001), estimated glomerular filtration rate (Chronic Kidney Disease Endemiology collaboration: r = −0.44; P < .0001; Modification of Diet in Renal Disease: r = −0.43; P < .001; Cockcroft-Gault r = −0.39; P < .01), serum phosphate (r = 0.34; P < .05). Upon multiple regression analysis renalase was predicted by 70% using age (beta value 0.21, P = 0.043), time after transplantation (beta value, 0.22; P = .037), serum creatinine (beta value, 0.50; P = .016), and diastolic blood pressure (beta value, 0.33; P = .027).

Conclusions

Renalase is highly elevated in kidney transplant recipients, predominantly dependent on kidney function, which deteriorates with time after kidney transplantation and age. Further studies are needed to establish its putative role in the pathogenesis of hypertension after transplantation and possible novel targeted therapies.     

Evidence from glut2-null mice that glucose is a critical physiological regulator of feeding

A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.     

Pdlim2 is a novel actin-regulating protein of podocyte foot processes

The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, α-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.