Biologically-coupled bisphosphonate chaperones effectively deliver molecules to the site of soft tissue-bone healing.

Tendon injuries are common and often treated surgically, however, current tendon repair healing results in poorly organized fibrotic tissue. While certain growth factors have been reported to improve both the strength and organization of the repaired enthesis, their clinical applicability is severely limited due to a lack of appropriate delivery strategies. In this study, we evaluated a recently developed fluorescent probe, Osteoadsorptive Fluorogenic Sentinel-3 that is composed of a bone-targeting bisphosphonate (BP) moiety linked to fluorochrome and quencher molecules joined via a cathepsin K-sensitive peptide sequence. Using a murine Achilles tendon-to-bone repair model, BP-based and/or Ctsk-coupled imaging probes were applied either locally or systemically. Fluorescence imaging was used to quantify the resultant signal in vivo. After tendon-bone repair, animals that received either local or systemic administration of imaging probes demonstrated significantly higher fluorescence signal at the repair site compared to the sham surgery group at all time points (p < 0.001), with signal peaking at 7-10 days after surgery. Our findings demonstrate the feasibility of using a novel BP-based targeting and Ctsk-activated delivery of molecules to the site of tendon-to-bone repair and creates a foundation for further development of this platform as an effective strategy to deliver bioactive molecules to sites of musculoskeletal injury.

CCN1/Cyr61 Is Required in Osteoblasts for Responsiveness to the Anabolic Activity of PTH.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvß3 and/or αvß5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvß3/ß5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvß3/ß5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin, resulting in pathologic cartilage and bone matrix deposition. Cyr61, CTGF, Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways, such as the STAT3 and PCP signaling pathways, that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.

Acute infectious laryngitis: A case series.

Although acute laryngitis is common, it is often managed by primary physicians. Therefore, video images documenting its signs are scarce. This series includes 7 professional voice users who previously had undergone baseline strobovideolaryngscopy (SVL) during routine examinations or during evaluations for other complaints and who returned with acute laryngitis. Sequential SVL showed not only the expected erythema, edema, cough, and dysphonia, but also new masses in 5 of the 7 subjects. All the signs returned to baseline. This series is reported to highlight the reversible structural changes that can be expected in patients with acute laryngitis and the value of conservative management.

CYR61/CCN1 Regulates Sclerostin Levels and Bone Maintenance.

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.

Fibrosis and hypoxia-inducible factor-1α-dependent tumors of the soft tissue on loss of von Hippel-Lindau in mesenchymal progenitors.

The hypoxia-inducible factor (Hif)-1α (Hif-1α) and Hif-2α (Epas1) have a critical role in both normal development and cancer. von Hippel Lindau (Vhl) protein, encoded by a tumor suppressor gene, is an E3 ubiquitin ligase that targets Hif-1α and Epas1 to the proteasome for degradation. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Human tumors of the soft tissue are a very complex and heterogeneous group of neoplasias. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling.

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1(-/-)) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1(-/-) mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1(-/-) mice had decreased trabecular bone volume/total volume and that both male and female Wisp1(-/-) mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1(-/-) mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1(-/-) mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1(-/-) mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1(-/-) bone marrow stromal cells had reduced expression of ß-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1(-/-) mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones.

CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts.

Many studies have reported that CCN family protein 2 (also known as connective tissue growth factor) induces fibrotic response in skeletal muscle, thus emphasizing the pathological role of CCN2 in muscle tissues. However, the physiological role of CCN2 in myogenesis is still unknown. This study clarified the CCN2 functions during myogenesis. Recombinant CCN2 (rCCN2) promoted proliferation and MyoD production in C2C12 cells and primary myoblasts, but inhibited myogenin production. In accordance with these findings, the gene expression levels of myosin heavy chain, which is a marker of terminally differentiated myoblasts and desmin, which is the main intermediate filament protein of muscle cells, were decreased by rCCN2 treatment. In vivo analyses with Ccn2-deficient skeletal muscle revealed decreased proliferating cell nuclear antigen (PCNA)/MyoD double positive cells and muscle hypoplasia. Consistent with this finding, myogenic marker genes and myotube formation were repressed in Ccn2-deficient myoblasts. The protein production of CCN2 was increased in C2C12 myoblasts treated with tumor necrosis factor-α, which is a pro-inflammatory cytokine, suggesting its role in muscle regeneration after inflammation. These findings indicate that CCN2 promotes proliferation and early differentiation but inhibits the terminal differentiation of myoblasts, thus suggesting that CCN2 plays a physiological role in myogenesis.

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis.

Bent bone dysplasia-FGFR2 type, a distinct skeletal disorder, has deficient canonical FGF signaling.

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.

CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis.

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor ß (TGFß) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.

Connective tissue growth factor is required for normal follicle development and ovulation.

Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-ß superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-ß family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-ß and progesterone signaling cascades and is necessary for normal follicle development and ovulation.

Roles for CCN2 in normal physiological processes.

CCN2, also known as connective tissue growth factor, is a member of the CCN (CCN1-6) family of modular matricellular proteins. Analysis of CCN2 function in vivo has focused primarily on its key role as a mediator of excess ECM synthesis in multiple fibrotic diseases. However, CCN2 and related family members are widely expressed during development. Recent studies using new genetic models are revealing that CCN2 has essential roles in the development of many tissues. This review focuses on current and emerging data on CCN2 and its functions in chondrogenesis and angiogenesis, and on new studies showing that CCN2 has essential functions during embryonic and postnatal development in a number of epithelial tissues.

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Systems genetics analysis of mouse chondrocyte differentiation.

One of the goals of systems genetics is the reconstruction of gene networks that underlie key processes in development and disease. To identify cartilage gene networks that play an important role in bone development, we used a systems genetics approach that integrated microarray gene expression profiles from cartilage and bone phenotypic data from two sets of recombinant inbred strains. Microarray profiles generated from isolated chondrocytes were used to generate weighted gene coexpression networks. This analysis resulted in the identification of subnetworks (modules) of coexpressed genes that then were examined for relationships with bone geometry and density. One module exhibited significant correlation with femur length (r = 0.416), anteroposterior diameter (r = 0.418), mediolateral diameter (r = 0.576), and bone mineral density (r = 0.475). Highly connected genes (n = 28) from this and other modules were tested in vitro using prechondrocyte ATDC5 cells and RNA interference. Five of the 28 genes were found to play a role in chondrocyte differentiation. Two of these, Hspd1 and Cdkn1a, were known previously to function in chondrocyte development, whereas the other three, Bhlhb9, Cugbp1, and Spcs3, are novel genes. Our integrative analysis provided a systems-level view of cartilage development and identified genes that may be involved in bone development.

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell-specific transmembrane protein (DC-STAMP).

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF-κB ligand (GST-RANKL), and a combination of recombinant CCN2 (rCCN2) and GST-RANKL significantly enhanced tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cell formation compared with GST-RANKL alone. Therefore, we suspected the involvement of CCN2 in cell-cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real-time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid-phase binding assay of CCN2 and dendritic cell-specific transmembrane protein (DC-STAMP), which is involved in cell-cell fusion. The results showed that CCN2 induced and interacted with DC-STAMP. Furthermore, GST-RANKL-induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC-STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC-STAMP.

Wnt10b deficiency results in age-dependent loss of bone mass and progressive reduction of mesenchymal progenitor cells.

Wnt10b is a canonical Wnt ligand expressed in developing bone and has been linked to mesenchymal progenitor functions in mice and humans. Because Wnt signaling has been shown to play an important role in progenitor maintenance in a variety of adult tissues, we examined bone deposition and growth rates throughout postnatal development in Wnt10b-null mice. Using bone histomorphometry and micro-computed tomographic (µCT) studies, we demonstrate that trabecular bone deposition is slightly enhanced in Wnt10b-null mice at 1 month of age, followed by progressive loss with age. Importantly, we find that Wnt10b is required for maintenance of adult bone density in multiple backgrounds of inbred mice and that both copies of the Wnt10b gene are required to maintain normal bone density in 6-month-old animals. We go on to show that the loss in trabecular bone in Wnt10b-null mice is associated with a reduction in the number of bone marrow-derived mesenchymal progenitors (MPCs) using in vitro colony-forming unit assays and marker analysis. Analysis of osteogenic gene expression in primary bone marrow stromal cells demonstrated reductions in expression of several osteoblast differentiation markers. Taken together, our results indicate that Wnt10b is uniquely required for maintenance of mesenchymal progenitor activity in adult bone. The results show the significance of studying individual Wnt ligands and their potentially unique contribution in the context of aging and disease.

Granulosa cell-expressed BMPR1A and BMPR1B have unique functions in regulating fertility but act redundantly to suppress ovarian tumor development.

Bone morphogenetic proteins (BMPs) have diverse roles in development and reproduction. Although several BMPs are produced by oocytes, thecal cells, and granulosa cells of developing follicles, the in vivo functions of most of these ligands are unknown. BMP signals are transduced by multiple type I and type II TGFbeta family receptors, and of the type I receptors, BMP receptor 1A (BMPR1A) and BMP receptor 1B (BMPR1B) are known to be expressed in rodent granulosa cells. Female mice homozygous null for Bmpr1b are sterile due to compromised cumulus expansion, but the function of BMPR1A in the ovary is unknown. To further decipher a role for BMP signaling in mouse granulosa cells, we deleted Bmpr1a in the granulosa cells of the ovary and found Bmpr1a conditional knockout females to be subfertile with reduced spontaneous ovulation. To explore the redundant functions of BMP receptor signaling in the ovary, we generated Bmpr1a Bmpr1b double-mutant mice, which developed granulosa cell tumors that have evidence of increased TGFbeta and hedgehog signaling. Thus, similar to SMAD1 and SMAD5, which have redundant roles in suppressing granulosa cell tumor development in mice, two type I BMP receptors, BMPR1A and BMPR1B, function together to prevent ovarian tumorigenesis. These studies support a role for a functional BMP signaling axis as a tumor suppressor pathway in the ovary, with BMPR1A and BMPR1B acting downstream of BMP ligands and upstream of BMP receptor SMADs.

BMP signaling and podocyte markers are decreased in human diabetic nephropathy in association with CTGF overexpression.

Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF(+/+) and CTGF(+/-) mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF(+/-) mice, pSmad1/5/8 was preserved, compared with diabetic CTGF(+/+) mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1.

BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation.

Bone morphogenetic protein (BMP) signaling is required for endochondral bone formation. However, whether or not the effects of BMPs are mediated via canonical Smad pathways or through noncanonical pathways is unknown. In this study we have determined the role of receptor Smads 1, 5 and 8 in chondrogenesis. Deletion of individual Smads results in viable and fertile mice. Combined loss of Smads 1, 5 and 8, however, results in severe chondrodysplasia. Smad1/5(CKO) (cartilage-specific knockout) mutant mice are nearly identical to Smad1/5(CKO);Smad8(-/-) mutants, indicating that Smads 1 and 5 have overlapping functions and are more important than Smad8 in cartilage. The Smad1/5(CKO) phenotype is more severe than that of Smad4(CKO) mice, challenging the dogma, at least in chondrocytes, that Smad4 is required to mediate Smad signaling through BMP pathways. The chondrodysplasia in Smad1/5(CKO) mice is accompanied by imbalances in cross-talk between the BMP, FGF and Ihh/PTHrP pathways. We show that Ihh is a direct target of BMP pathways in chondrocytes, and that FGF exerts antagonistic effects on Ihh expression. Finally, we tested whether FGF exerts its antagonistic effects directly through Smad linker phosphorylation. The results support the alternative conclusion that the effects of FGFs on BMP signaling are indirect in vivo.