Expression and localisation of RXFP3 in human spermatozoa and impact of INSL7 on sperm functions

Insulin‐like peptide 7 (INSL7) or relaxin‐3 is a member of the insulin superfamily that is recently discovered. This hormone interacts with relaxin family peptide receptor 3 (RXFP3). Although recent studies of INSL7 have focused on its function in the brain as a neuropeptide, spermatozoa may be a candidate target of INSL7 due to its detection in testes and contains binding sites. Therefore, this study aims to analyse the expression and localisation of RXFP3 on human spermatozoa and to assess the effect of INSL7 on human sperm motility. We have incubated normal semen samples in different doses of INSL7. Sperm motility was analysed by Computer Assisted Sperm Analysis. Moreover, localisation and expression of RXFP3 were assessed in human spermatozoa by immunofluorescence and RT‐PCR respectively. This study indicated that RXFP3 mainly localised in the post‐acrosomal region of sperm head and neck. However, we did not observe expression of RXFP3 mRNA in human spermatozoa. This study showed that INSL7 alleviated the natural decline in sperm motility after a 4‐hr incubation period. This was particularly observed in the 1.8 pmol/L treated samples. These data suggested that most likely expression of RXFP3 arrested in spermiogenesis, but the RXFP3 peptide existed on the surface of mature spermatozoa.     

Exendin‐4 shows no effects on the prostatic index in high‐fat‐diet‐fed rat with benign prostatic hyperplasia by improving insulin resistance

Benign prostatic hyperplasia (BPH) is a prevalent disease globally, and accumulating evidence has indicated an association between BPH, insulin resistance (IR) and diabetes. Exendin‐4 is widely used in clinics, which could enhance the proliferation of pancreatic β cells. The ability of exendin‐4 to promote tumorigenesis has been of concern, and whether exendin‐4 would enhance the propagation of BPH is not fully understood. We aimed to determine whether glucagon‐like peptide‐1 receptors (GLP‐1Rs) were expressed in rat prostate and to determine the effect of exendin‐4 on prostate of BPH. Male Wistar rats were used and assigned to six groups: normal diet (ND), high‐fat diet (HFD), HFD + exendin‐4, HFD + BPH, HFD + BPH + exendin‐4 and HFD + BPH + rosiglitazone group. After castration, steroids were injected subcutaneously for 4 weeks to induce BPH. Rats were kept on high‐fat diet to induce IR. Treatment groups were treated with exendin‐4 and rosiglitazone. Prostatic index and HOMA‐IR index were used to evaluate the prostatic hyperplasia status and the degree of IR respectively. The expression of GLP‐1R was indicated not only by immunohistochemistry, but also by Western blot analysis. The expression of GLP‐1R was significantly higher, and HOMA‐IR index and body weight significantly decreased after administration of exendin‐4. However, no significant differences in the prostatic index were observed between exendin‐4 treatment groups and non‐exendin‐4 treatment groups. Prostatic index was not influenced by exendin‐4 maybe by improving IR and weight loss.     

Thyroid hormone‐mediated growth and differentiation of growth plate chondrocytes involves IGF‐1 modulation of β‐catenin signaling

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β‐catenin signaling pathway. Insulin‐like growth factor 1 (IGF‐1) has been described as a stabilizer of β‐catenin, and thyroid hormone is a known stimulator of IGF‐1 receptor expression. The purpose of this study was to test the hypothesis that IGF‐1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β‐catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF‐1 and the IGF‐ receptor (IGF1R) stimulate Wnt‐4 expression and β‐catenin activation in growth plate chondrocytes. The positive effects of IGF‐1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T₃ activates IGF‐1/IGF1R signaling and IGF‐1‐dependent PI3K/Akt/GSK‐3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T₃‐mediated Wnt‐4 expression, β‐catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β‐catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF‐1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF‐1/IGF1R‐mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β‐catenin signaling, which is at least in part initiated by IGF‐1 signaling or the IGF‐1‐activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research     

Genetic analysis of the human Insulin‐like 3 gene: absence of mutations in a Greek paediatric cohort with testicular maldescent

This study investigated the hypothesis that genetic alterations of the human insulin‐like 3 (INSL3) gene are associated with testicular maldescent (TMD). Genomic DNA was extracted and amplified from peripheral blood samples of 170 unrelated children with all possible phenotypical expressions of TMD and 50 volunteers with normal external genitalia from the general paediatric population (controls). PCR‐single strand conformation polymorphism analysis was used to screen INSL3 gene for genetic variants. For rapid screening of a detected nonsilent genetic alteration, restriction assay using endonuclease Eag I was further employed. Products were analysed on 2% agarose gel and restriction patterns were visualised by ethidium bromide. Differences in genotype and allelic distributions of nonsilent genetic alterations were evaluated between (i) patients–controls, (ii) familial–sporadic, (iii) bilateral–unilateral and (iv) intra‐abdominal–inguinal cases of TMD. No mutations were detected. Three common INSL3 gene polymorphisms (27G>A, 126G>A, 178G>A) unrelated to any particular phenotype of TMD were detected both in patients and controls. These results indicate that INSL3 gene mutations are not a common cause of TMD in the human.     

Protection From Glucocorticoid‐Induced Osteoporosis by Anti‐Catabolic Signaling in the Absence of Sost/Sclerostin

Excess of glucocorticoids, either due to disease or iatrogenic, increases bone resorption and decreases bone formation and is a leading cause of osteoporosis and bone fractures worldwide. Improved therapeutic strategies are sorely needed. We investigated whether activating Wnt/β‐catenin signaling protects against the skeletal actions of glucocorticoids, using female mice lacking the Wnt/β‐catenin antagonist and bone formation inhibitor Sost. Glucocorticoids decreased the mass, deteriorated the microarchitecture, and reduced the structural and material strength of bone in wild‐type (WT), but not in Sost^–/– mice. The high bone mass exhibited by Sost^–/– mice is due to increased bone formation with unchanged resorption. However, unexpectedly, preservation of bone mass and strength in Sost^–/– mice was due to prevention of glucocorticoid‐induced bone resorption and not to restoration of bone formation. In WT mice, glucocorticoids increased the expression of Sost and the number of sclerostin‐positive osteocytes, and altered the molecular signature of the Wnt/β‐catenin pathway by decreasing the expression of genes associated with both anti‐catabolism, including osteoprotegerin (OPG), and anabolism/survival, such as cyclin D1. In contrast in Sost^–/– mice, glucocorticoids did not decrease OPG but still reduced cyclin D1. Thus, in the context of glucocorticoid excess, activation of Wnt/β‐catenin signaling by Sost/sclerostin deficiency sustains bone integrity by opposing bone catabolism despite markedly reduced bone formation and increased apoptosis. This crosstalk between glucocorticoids and Wnt/β‐catenin signaling could be exploited therapeutically to halt resorption and bone loss induced by glucocorticoids and to inhibit the exaggerated bone formation in diseases of unwanted hyperactivation of Wnt/β‐catenin signaling. © 2016 American Society for Bone and Mineral Research.     

IL‐36 Induces Bisphosphonate‐Related Osteonecrosis of the Jaw‐Like Lesions in Mice by Inhibiting TGF‐β‐Mediated Collagen Expression

Long‐term administration of nitrogen‐containing bisphosphonates can induce detrimental side effects such as bisphosphonate‐related osteonecrosis of the jaw (BRONJ) in human. Although inflammation is known to be associated with BRONJ development, the detailed underlying mechanism remains unknown. Here, we report that the pro‐inflammatory cytokine IL‐36α is, in part, responsible for the BRONJ development. We found a notably higher level of IL‐36α and lower level of collagen in the BRONJ lesions in mice. We also found that IL‐36α remarkably suppressed TGF‐β‐mediated expression of Collα1 and α‐Sma via the activation of Erk signaling pathway in mouse gingival mesenchymal stem cells. When IL‐36 signaling was abrogated in vivo, development of BRONJ lesions was ameliorated in mice. Taken together, we showed the pathologic role of IL‐36α in BRONJ development by inhibiting collagen expression and demonstrated that IL‐36α could be a potential marker and a therapeutic target for the prevention and treatment of BRONJ. © 2016 American Society for Bone and Mineral Research.     

Finite Element Analysis Based on In Vivo HR‐pQCT Images of the Distal Radius Is Associated With Wrist Fracture in Postmenopausal Women

BMD, bone microarchitecture, and bone mechanical properties assessed in vivo by finite element analysis were associated with wrist fracture in postmenopausal women. Introduction: Many fractures occur in individuals with normal BMD. Assessment of bone mechanical properties by finite element analysis (FEA) may improve identification of those at high risk for fracture. Materials and Methods: We used HR‐pQCT to assess volumetric bone density, microarchitecture, and μFE‐derived bone mechanical properties at the radius in 33 postmenopausal women with a prior history of fragility wrist fracture and 33 age‐matched controls from the OFELY cohort. Radius areal BMD (aBMD) was also measured by DXA. Associations between density, microarchitecture, mechanical parameters and fracture status were evaluated by univariate logistic regression analysis and expressed as ORs (with 95% CIs) per SD change. We also conducted a principal components (PCs) analysis (PCA) to reduce the number of parameters and study their association (OR) with wrist fracture. Results: Areal and volumetric densities, cortical thickness, trabecular number, and mechanical parameters such as estimated failure load, stiffness, and the proportion of load carried by the trabecular bone at the distal and proximal sites were associated with wrist fracture (p < 0.05). The PCA revealed five independent components that jointly explained 86.2% of the total variability of bone characteristics. The first PC included FE‐estimated failure load, areal and volumetric BMD, and cortical thickness, explaining 51% of the variance with an OR for wrist fracture = 2.49 (95% CI, 1.32–4.72). Remaining PCs did not include any density parameters. The second PC included trabecular architecture, explaining 12% of the variance, with an OR = 1.82 (95% CI, 0.94–3.52). The third PC included the proportion of the load carried by cortical versus trabecular bone, assessed by FEA, explaining 9% of the variance, and had an OR = 1.61 (95% CI, 0.94–2.77). Thus, the proportion of load carried by cortical versus trabecular bone seems to be associated with wrist fracture independently of BMD and microarchitecture (included in the first and second PC, respectively). Conclusions: These results suggest that bone mechanical properties assessed by μFE may provide information about skeletal fragility and fracture risk not assessed by BMD or architecture measurements alone and are therefore likely to enhance the prediction of wrist fracture risk.     

Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF‐β signaling

Transforming growth factor‐β (TGF‐β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF‐β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF‐β during the whole time course of osteoarthritis is important. To investigate the activity of TGF‐β in osteoarthritis progression, we collected knee joints from Dunkin–Hartley (DH) guinea pigs at 3, 6, 9, and 12‐month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro‐computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis, and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF‐β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age‐related changes of osterix⁺ osteoprogenitors were in parallel with active TGF‐β, which implied the excessive osteogenesis may link to the activity of TGF‐β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF‐β signaling in osteoarthritis progression of DH guinea pigs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:763–770, 2016.     

Repeated Immobilization Stress Increases Expression of β3‐Adrenoceptor in the Left Ventricle and Atrium of the Rat Heart

Stress is a contributor of many cardiovascular diseases. Positive inotropic and chronotropic effects of catecholamines are regulated via β‐adrenergic receptors (ARs). Many reports exist concerning changes of cardiac β₁‐ and β₂‐ARs in stress, but only a few deal with modulation of cardiac β₃‐AR. Our aim was to analyze the expression and binding sites of β₁‐, β₂‐ and β₃‐ARs and adenylyl cyclase activity in the left ventricle, and β₃‐AR expression and binding in the left atrium of rats exposed to acute and chronic immobilization stress (IMO). The concentration of noradrenaline in the ventricle decreased, while adrenaline increased, especially after repeated IMO. The mRNA and protein levels, and binding sites of β₃‐subtype significantly rose following chronic IMO, while all parameters for β₂‐AR dropped after single and repeated exposure. Similarly, the mRNA levels and binding sites for β₃‐subtype increased in the left atrium as a consequence of chronic IMO. The rise in β₃‐subtypes and a drop in β₂‐subtypes resulted in inhibition of adenylyl cyclase activity within the left ventricle. Taken together, among other factors, up‐regulation of β₃‐AR could represent an adaptation mechanism, which might be related to altered physiological function of the left ventricle and atrium during prolonged emotional stress and might serve cardioprotective function during catecholamine overload. Copyright © 2013 John Wiley & Sons, Ltd.     

Expression of hypoxia‐inducible factor‐1α and hepatocyte growth factor in development of fibrosis in the transplanted kidney

Late renal graft loss is associated with interstitial fibrosis. Hypoxia‐inducible factor‐1α (HIF‐1α) is thought to facilitate fibrosis through interaction with TGF‐β1, while hepatocyte growth factor (HGF) may act antifibrotic in the kidney allograft. The aim of this study was to investigate the expression of HIF‐1α and HGF in protocol biopsies as possible prognostic biomarkers for renal fibrosis. Thirty‐nine renal transplant recipients were included in the study. Protocol biopsies performed 1 and 2 years after transplantation were used for immunohistochemistry analysis. The correlation between HIF‐1α/HGF and the Banff score was analysed. In addition, progression in renal fibrosis and graft survival among recipients with high or low expression of HIF‐1α/HGF after transplantation was compared. There was no significant correlation between fibrosis and the HIF‐1α expression 1 and 2 years after transplantation, but an inverse significant correlation between the HGF expression and the fibrosis score 1 year after transplantation was shown. Even when adjusting for human leucocyte antigen mismatches, there was a significant relationship between fibrosis and HGF expression. Graft survival was not significantly correlated to HIF‐1α or HGF at 1 year, although the trend was towards better graft survival with high HGF. HGF may have antifibrotic effects in human renal transplants. (Central.Denmark.Region.Committee number: 1‐10‐72‐318‐13)     

−509C>T polymorphism in the TGF‐β1 gene promoter is not associated with susceptibility to and progression of colorectal cancer in Chinese

Aim Colorectal cancer is common, accounting for nearly 10% of all cancers. Transforming growth factor‐β1 (TGF‐β1) is a pleiotropic cytokine that has been implicated in the pathogenesis of colorectal neoplasia. The most studied −509C>T polymorphism of TGF‐β1 gene has been associated with various kinds of cancer. This study investigated the association between this genetic variant and the risk and/or progression of colorectal cancer. Method A case–control study was carried out of 150 colorectal cancer cases and 503 healthy controls. DNA was extracted from blood cell nuclear materials, and −509C>T polymorphism in the TGF‐β1 gene promoter was genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Colorectal cancer tissues (n = 70) were obtained from the studied cases for measurement of TGF‐β1 mRNA expression levels. We also assessed the plasma TGF‐β1 levels of cases (n = 88) and healthy subjects (n = 120). Results The TGF‐β1 producer genotype, −509TT, was not associated with an increased risk of colorectal cancer compared with other genotypes. Colorectal cancer patients especially those with a more aggressive disease behaviour were more frequently associated with C allele. Conclusion The results suggest that TGF‐β1 −509C>T polymorphism is not associated with either an increased risk or progression of colorectal cancer.     

Subcellular distribution of the insulin‐like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin‐like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF‐binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP‐2 and IGFBP‐3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP‐2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP‐2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP‐2 were in the cytoplasmic fraction. Immunoreactive IGFBP‐3 was found in the cytoplasmic and peri‐nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP‐2 and IGFBP‐3 but only IGFBP‐3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1421–1427, 2008     

Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence,Genotype/Phenotype Correlation,and a Novel Non‐UBA Domain Mutation (P364S) Associated With Increased NF‐κB Signaling Without Loss of Ubiquitin Binding

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin‐associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co‐transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF‐κB luciferase reporter gene. GST‐SQSTM1 (wildtype and mutant) proteins were used in pull‐down assays to compare monoubiquitin‐binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF‐κB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF‐κB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.