Parathyroid hormone (PTH) regulates calcium homeostasis and bone metabolism by activating PTH type I receptor (PTH1R). Here we show that transforming growth factor (TGF)-β type II receptor (TβRII) forms an endocytic complex with PTH1R in response to PTH and regulates signalling by PTH and TGF-β. TβRII directly phosphorylates the PTH1R cytoplasmic domain, which modulates PTH-induced endocytosis of the PTH1R–TβRII complex. Deletion of TβRII in osteoblasts increases the cell-surface expression of PTH1R and augments PTH signalling. Conditional knockout of TβRII in osteoblasts in mice results in a high bone mass with increased trabecular bone and decreased cortical bone, similar to the bone phenotype in mice expressing a constitutively active PTH1R. Disruption of PTH signalling by injection of PTH(7–34) or ablation of PTH1R rescues the bone phenotype of TβRII knockout mice. These studies reveal a previously unrecognized function for TβRII and a mechanism for integration of PTH and local growth factor at the membrane receptor level.

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BackgroundAmong the largest and most diverse transcription factor families in plants, basic leucine zipper (bZIP) family participate in regulating various processes, including floral induction and development, stress and hormone signaling, photomorphogenesis, seed maturation and germination, and pathogen defense. Although common wheat (Triticum aestivum L.) is one of the most widely cultivated and consumed food crops in the world, there is no comprehensive analysis of bZIPs in wheat, especially those involved in anther development. Previous studies have demonstrated wheat, T. urartu, Ae. tauschii, barley and Brachypodium are evolutionarily close in Gramineae family, however, the real evolutionary relationship still remains mysterious.ResultsIn this study, 187 bZIP family genes were comprehensively identified from current wheat genome. 98, 96 and 107 members of bZIP family were also identified from the genomes of T.urartu, Ae.tauschii and barley, respectively. Orthology analyses suggested 69.4 % of TubZIPs were orthologous to 68.8 % of AetbZIPs and wheat had many more in-paralogs in the bZIP family than its relatives. It was deduced wheat had a closer phylogenetic relationship with barley and Brachypodium than T.urartu and Ae.tauschii. bZIP proteins in wheat, T.urartu and Ae.tauschii were divided into 14 subgroups based on phylogenetic analyses. Using Affymetrix microarray data, 48 differentially expressed TabZIP genes were identified to be related to anther development from comparison between the male sterility line and the restorer line. Genes with close evolutionary relationship tended to share similar gene structures. 15 of 23 selected TabZIP genes contained LTR elements in their promoter regions. Expression of 21 among these 23 TabZIP genes were obviously responsive to low temperature. These 23 TabZIP genes all exhibited distinct tissue-specific expression pattern. Among them, 11 TabZIP genes were predominantly expressed in anther and most of them showed over-dominance expression mode in the cross combination TY806 × BS366.ConclusionsThe genome-wide identification provided an overall insight of bZIP gene family in wheat and its relatives. The evolutionary relationship of wheat and its relatives was proposed based on orthology analyses. Microarray and expression analyses suggested the potential involvement of bZIP genes in anther development and facilitated selection of anther development related gene for further functional characterization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2196-7) contains supplementary material, which is available to authorized users.

Ligand binding to certain heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) stimulates the rapid synthesis of cyclic adenosine monophosphate (cAMP) through the G protein αs subunit, which activates adenylyl cyclase (AC). We found that the transmembrane receptor low-density lipoprotein receptor–related protein 6 (LRP6), a co-receptor for Wnt proteins, bound to the Gαsβγ heterotrimer and that knockdown of LRP6 attenuated cAMP production by various GPCRs, including parathyroid hormone receptor 1 (PTH1R). Knockdown of LRP6 disrupted the localization of Gαs to the plasma membrane, which led to a decrease in the extent of coupling of Gαs to PTH1R and inhibited the production of cAMP and the activation of cAMP-dependent protein kinase (PKA) in response to PTH. PKA phosphorylated LRP6, which enhanced the binding of Gαs to LRP6, its localization to the plasma membrane, and the production of cAMP in response to PTH. Decreased PTH-dependent cAMP production was observed in single cells in which LRP6 was knocked down or mutated at the PKA site by monitoring the cAMP kinetics. Thus, we suggest that the binding of Gαs to LRP6 is required to establish a functional GPCR-Gαs-AC signaling pathway for the production of cAMP, providing an additional regulatory component to the current GPCR-cAMP paradigm.

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Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10−6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks pos...

Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome-associated FYVE-domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein-phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast-enhanced mCT imaging of Microfil-perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis.

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.