Characterization of metabolic health in mouse models of fibrillin-1 perturbation

Mutations in the microfibrillar protein fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFβ signaling due to an inability to sequester latent or active forms of TGFβ, respectively. Mouse models of excess TGFβ signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which fibrillin-1 mutation leads to aberrant TGFβ signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple fibrillin-1 mutant mouse strains to understand how fibrillin-1 contributes to metabolic health. The results of this study demonstrate that fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not fibrillin-1 mutant mice had elevated TGFβ signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of fibrillin in some tissues.     

Anti-transforming growth factor ß antibody treatment rescues bone loss and prevents breast cancer metastasis to bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p?=?0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p?=?0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.     

Fascin protein is critical for transforming growth factor β protein-induced invasion and filopodia formation in spindle-shaped tumor cells

Fascin, an actin-bundling protein overexpressed in all carcinomas, has been associated with poor prognosis, shorter survival, and more metastatic diseases. It is believed that fascin facilitates tumor metastasis by promoting the formation of invasive membrane protrusions. However, the mechanisms by which fascin is overexpressed in tumors are not clear. TGFβ is a cytokine secreted by tumor and mesenchymal cells and promotes metastasis in many late stage tumors. The pro-metastasis mechanisms of TGFβ remain to be fully elucidated. Here we demonstrated that TGFβ induced fascin expression in spindle-shaped tumor cells through the canonical Smad-dependent pathway. Fascin was critical for TGFβ-promoted filopodia formation, migration, and invasion in spindle tumor cells. More importantly, fascin expression significantly correlates with TGFβ1 and TGFβ receptor I levels in a cohort of primary breast tumor samples. Our results indicate that elevated TGFβ level in the tumor microenvironment may be responsible for fascin overexpression in some of the metastatic tumors. Our data also suggest that fascin could play a central role in TGFβ-promoted tumor metastasis.     

Inhibition of induced endochondral bone development in caffeine-treated rats

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGFβ were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.     

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of α-smooth muscle (α-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, α-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor β (TGFβ) in FBS and PCS was determined and the effect of neutralizing anti-TGFβ antibody was evaluated. It was determined that FBS contained more TGFβ than PCS, but that essentially all the TGFβ was latent in both sera. However, results obtained with anti-TGFβ antibody show that active TGFβ is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFβ or an enzyme that is able to activate latent serum TGFβ. Alternatively, sera may contain two different forms of latent TGFβ, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds. J. Cell. Physiol. 171:1–10, 1997. © 1997 Wiley-Liss, Inc.     

TGFβ and BMP signaling in cardiac cushion formation: lessons from mice and chicken

Cardiac cushion formation is crucial for both valvular and septal development. Disruption in this process can lead to valvular and septal malformations, which constitute the largest part of congenital heart defects. One of the signaling pathways that is important for cushion formation is the TGFβ superfamily. The involvement of TGFβ and BMP signaling pathways in cardiac cushion formation has been intensively studied using chicken in vitro explant assays and in genetically modified mice. In this review, we will summarize and discuss the role of TGFβ and BMP signaling components in cardiac cushion formation.     

Follistatin as a potent regulator of bone metabolism

Follistatin is a monomeric glycoprotein, distributed in a wide range of tissues. Recent work has demonstrated that this protein is a pluripotential molecule that has no structural similarity but is functionally associated with members of the transforming growth factor (TGF)-β superfamily, which indicates its wide range of action. Members of the TGF-β superfamily, especially activins and bone morphogenetic proteins are involved in bone metabolism. They play an important role in bone physiology, influencing bone growth, turnover, bone formation and cartilage induction. As follistatin is considered to be the antagonist of the TGF-β superfamily members, it plays an important role in bone metabolism and development.     

The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways

The members of the transforming growth factor beta (TGFβ) family of cytokines, including bone morphogenetic proteins (BMP), play fundamental roles in development and tissue homeostasis. Hence, aberrant TGFβ/BMP signalling is associated with several human diseases such as fibrosis, bone and immune disorders, cancer progression and metastasis. Consequently, targeting TGFβ signalling for intervention potentially offers therapeutic opportunities against these diseases. Many investigations have focussed on understanding the molecular mechanisms underpinning the regulation of TGFβ signalling. One of the key areas has been to investigate the regulation of the protein components of the TGFβ/BMP signal transduction pathways by ubiquitylation and deubiquitylation. In the last 15 years, extensive research has led to the discovery and characterisation of several E3 ubiquitin ligases that influence the TGFβ pathway. However, the research on DUBs regulating the TGFβ pathway has received prominence only recently and is still an emerging field. This review will provide a concise summary of our current understanding of how DUBs regulate TGFβ signalling.     

TGFβ signaling in cartilage development and maintenance

Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome‐wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage. Birth Defects Research (Part C) 102:37–51, 2014. © 2014 Wiley Periodicals, Inc.     

The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche

The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFβ family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvβ3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFβ signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.     

Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization

It is well established that 1α-25-dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre-osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon-related and -regulated genes (ISG) to be overrepresented in the set of 1,25D3-regulated genes. 1,25D3 down-regulated ISGs predominantly during the pre-mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ- and 1,25D3-mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti-correlated with interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis.     

Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures

Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

Smad7 regulates terminal maturation of chondrocytes in the growth plate

Members of the bone morphogenetic protein (BMP) superfamily, including transforming growth factor-betas (TGFβ), regulate multiple aspects of chondrogenesis. Smad7 is an intracellular inhibitor of BMP and TGFβ signaling. Studies in which Smad7 was overexpressed in chondrocytes demonstrated that Smad7 can impact chondrogenesis by inhibiting BMP signaling. However, whether Smad7 is actually required for endochondral ossification in vivo is unclear. Moreover, whether Smad7 regulates TGFβ in addition to BMP signaling in developing cartilage is unknown. In this study, we found that Smad7 is required for both axial and appendicular skeletal development. Loss of Smad7 led to impairment of the cell cycle in chondrocytes and to defects in terminal maturation. This phenotype was attributed to upregulation of both BMP and TGFβ signaling in Smad7 mutant growth plates. Moreover, Smad7−/− mice develop hypocellular cores in the medial growth plates, associated with elevated HIF1α levels, cell death, and intracellular retention of types II and X collagen. Thus, Smad7 may be required to mediate cell stress responses in the growth plate during development.     

Unchaining the beast; insights from structural and evolutionary studies on TGFβ secretion,sequestration, and activation

TGFβ is secreted in a latent state and must be “activated” by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFβ mobilisation in vivo. Here we review the process of TGFβ secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFβ is secreted, sequestered and released. This allows the separation of more ancient TGFβ activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.     

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.     

Cellular responses to TGFβ and TGFβ receptor expression in human colonic epithelial cells require CaSR expression and function

CaSR and TGFβ are robust promoters of differentiation in the colonic epithelium. Loss of cellular responses to TGFβ or loss of CaSR expression is tightly linked to malignant progression. Human colonic epithelial CBS cells, originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer a unique opportunity in determining the functional linkage (if any) between CaSR and TGFβ. Knocking down CaSR expression abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca²⁺ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca²⁺ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR; there was no up-regulation of TGFβ receptor. It is concluded that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function.