Relevant use of Klotho in FGF19 subfamily signaling system in vivo

α-Klotho (α-Kl) and its homolog, β-Klotho (β-Kl) are key regulators of mineral homeostasis and bile acid/cholesterol metabolism, respectively. FGF15/ humanFGF19, FGF21, and FGF23, members of the FGF19 subfamily, are believed to act as circulating metabolic regulators. Analyses of functional interactions between α- and β-Kl and FGF19 factors in wild-type, α-kl ^−/−, and β-kl ^−/− mice revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of α-Kl, FGF23, and 1,25(OH)₂D and an analogous regulatory network composed of β-Kl, FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Contrary to in vitro data, β-Kl is not essential for FGF21 signaling in adipose tissues in vivo, because (i) FGF21 signals are transduced in the absence of β-Kl, (ii) FGF21 could not be precipitated by β-Kl, and (iii) essential phenotypes in Fgf21 ^−/− mice (decreased expressions of Hsl and Atgl in WAT) were not replicated in β-kl ^−/− mice. These findings suggest the existence of Klotho-independent FGF21 signaling pathway(s) where undefined cofactors are involved. One-to-one functional interactions such as α-Klotho/FGF23, β-Klotho/FGF15 (humanFGF19), and undefined cofactor/FGF21 would result in tissue-specific signal transduction of the FGF19 subfamily.     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Bone morphogenetic protein and transforming growth factor beta inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin-1beta

OBJECTIVE: Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta signaling, inhibitory Smad6 (I-Smad6) and I-Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin-1beta (IL-1beta) stimulation in vitro. METHODS: RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I-Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen. RESULTS: Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I-Smads was found. In cultured articular chondrocytes, stimulation with IL-1beta showed up-regulation of Smad7, whereas Smad6 was down-regulated. CONCLUSION: Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I-Smads in articular cartilage in vivo. No evidence was found that up-regulation or down-regulation of I-Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.     

Getting ‘Smad' about obesity and diabetes

Recent findings on the role of transforming growth factor (TGF)-β/Smad3 signaling in the pathogenesis of obesity and type 2 diabetes have underscored its importance in metabolism and adiposity. Indeed, elevated TGF-β has been previously reported in human adipose tissue during morbid obesity and diabetic neuropathy. In this review, we discuss the pleiotropic effects of TGF-β/Smad3 signaling on metabolism and energy homeostasis, all of which has an important part in the etiology and progression of obesity-linked diabetes; these include adipocyte differentiation, white to brown fat phenotypic transition, glucose and lipid metabolism, pancreatic function, insulin signaling, adipocytokine secretion, inflammation and reactive oxygen species production. We summarize the recent in vivo findings on the role of TGF-β/Smad3 signaling in metabolism based on the studies using Smad3^−/− mice. Based on the presence of a dual regulatory effect of Smad3 on peroxisome proliferator-activated receptor (PPAR)β/δ and PPARγ2 promoters, we propose a unifying mechanism by which this signaling pathway contributes to obesity and its associated diabetes. We also discuss how the inhibition of this signaling pathway has been implicated in the amelioration of many facets of metabolic syndromes, thereby offering novel therapeutic avenues for these metabolic conditions.     

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb^th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.     

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2^−/− mice are viable, fertile and phenotypically indistinguishable from FGF2^+/+ littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2^−/− mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor β/bone morphogenetic protein inhibitors during experimental osteoarthritis

Objective

Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.

Methods

We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.

Results

Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.

Conclusion

Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.

     

BMP inhibition initiates neural induction via FGF signaling and Zic genes

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun—unlike the other BMP inhibitors used previously—can trigger conversion of Xenopus epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3 and foxD5a. Conversely, we found that zic1 can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.     

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Objective

Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR‐1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods

FGFR‐1 was deleted from the articular chondrocytes of adult mice in a cartilage‐specific and tamoxifen‐inducible manner. Two OA models (aging‐associated spontaneous OA, and destabilization‐induced OA), as well as an antigen‐induced arthritis (AIA) model, were established and tested in Fgfr1‐deficient and wild‐type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR‐1 inhibitor on human articular chondrocytes was examined.

Results

The gross morphologic features of Fgfr1‐deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12‐month‐old Fgfr1‐deficient mice displayed greater aggrecan staining compared to 12‐month‐old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR‐1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP‐13) and up‐regulation of FGFR‐3 in mouse and human articular chondrocytes.

Conclusion

Disruption of FGFR‐1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down‐regulation of MMP‐13 expression and up‐regulation of FGFR‐3 levels may contribute to the phenotypic changes observed in Fgfr1‐deficient mice.

     

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor α chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn^−/−) and lyn-deficient (Lyn^−/−) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor α chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn^−/−) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn^−/− mice, and it was severely impaired in B cells from Lyn^−/− and Fyn/Lyn^−/− mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn^−/− and Fyn/Lyn^−/− mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein inhibitors during experimental osteoarthritis

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor beta (TGFbeta) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFbeta and BMP during papain-induced OA-like changes in mice. METHODS: We used adenoviral overexpression of TGFbeta and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan-specific TGFbeta antagonist called murine latency-associated peptide 1 (mLAP-1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFbeta/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss. RESULTS: Intraarticular injection of papain caused increased protein expression of several TGFbeta and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFbeta inhibitor mLAP-1 increased articular cartilage PG loss. CONCLUSION: Our results indicate a pivotal role of endogenous TGFbeta in the development of osteophytes and synovial thickening, implicating endogenous TGFbeta in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFbeta signifies the protective role of TGFbeta in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.     

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK α and ζ synergistically promote T cell maturation in the thymus. Absence of both DGKα and ζ (DGKα^−/−ζ^−/−) results in a severe decrease in the number of CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKα^−/−ζ^−/− mice. The developmental blockage in DGKα^−/−ζ^−/− mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.     

Protein tyrosine phosphatases PTP-1B and TC-PTP play nonredundant roles in macrophage development and IFN-γ signaling

The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp^−/−ptp1b^−/−) is lethal at day E9.5–10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B–deficient background (tcptp^+/−ptp1b^−/−) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-γ, as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition, splenic T cells demonstrated increased IFN-γ secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp^+/−ptp1b^+/−) exhibited normal development, confirming that these genes are not interchangeable. Together, these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling.     

Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,β-methyleneadenosine-5′-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.