Matrix-dependent perturbation of TGFβ signaling and disease

Transforming growth factor beta (TGFβ) is a multipotent cytokine that is sequestered in the extracellular matrix (ECM) through interactions with a number of ECM proteins. The ECM serves to concentrate latent TGFβ at sites of intended function, to influence the bioavailability and/or function of TGFβ activators, and perhaps to regulate the intrinsic performance of cell surface effectors of TGFβ signal propagation. The downstream consequences of TGFβ signaling cascades in turn provide feedback modulation of the ECM. This review covers recent examples of how genetic mutations in constituents of the ECM or TGFβ signaling cascade result in altered ECM homeostasis, cellular performance and ultimately disease, with an emphasis on emerging therapeutic strategies that seek to capitalize on this refined mechanistic understanding.     

TGFβ signaling and cardiovascular diseases

Transforming growth factor β (TGFβ) family members are involved in a wide range of diverse functions and play key roles in embryogenesis, development and tissue homeostasis. Perturbation of TGFβ signaling may lead to vascular and other diseases. In vitro studies have provided evidence that TGFβ family members have a wide range of diverse effects on vascular cells, which are highly dependent on cellular context. Consistent with these observations genetic studies in mice and humans showed that TGFβ family members have ambiguous effects on the function of the cardiovascular system. In this review we discuss the recent advances on TGFβ signaling in (cardio)vascular diseases, and describe the value of TGFβ signaling as both a disease marker and therapeutic target for (cardio)vascular diseases.     

Interaction of TGFβ and BMP signaling pathways during chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.     

Genomic organization, chromosomal mapping, and analysis of the 5’ promoter region of the human MAdCAM-1 gene

 MAdCAM-1, the endothelial addressin cell adhesion molecule-1, interacts preferentially with the leukocyte β7 integrin LPAM-1 (α4β7), but also with L-selectin, and with VLA-4 (α4β1) on myeloid cells, and serves to direct leukocytes into mucosal and inflamed tissues. Overlapping cosmid and phage λ genomic clones were isolated, revealing that the human MAdCAM-1 gene contains five exons where the signal peptide, two Ig domains, and mucin domain are each encoded by separate exons. The transmembrane domain, cytoplasmic domain, and 3′ untranslated region are encoded together on exon 5. The mucin domain contains eight repeats in total that are subject to alternative splicing. Despite the absence of a human counterpart of the third IgA-homologous domain and lack of sequence conservation of the mucin domain, the genomic organizations of the human and mouse MAdCAM-1 genes are similar. An alternatively spliced MAdCAM-1 variant was identified that lacks exon 4 encoding the mucin domain, and may mediate leukocyte adhesion to LPAM-1 without adhesion to the alternate receptor, L-selectin. The MAdCAM-1 gene was located at p13.3 on chromosome 19, in close proximity to the ICAM-1 and ICAM-3 genes (p13.2-p13.3). PMA-inducible promotor activity was contained in a 700 base pair 5’ flanking fragment conserved with the mouse MAdCAM-1 gene including tandem NF-kB sites, and an Sp1 site; and in addition multiple potential AP2, Adh1 (ETF), PEA3, and Sp1 sites. In summary, the data establish that the previously reported human MAdCAM-1 cDNA does indeed encode the human homologue of mouse MAdCAM-1, despite gross dissimilarities in the MAdCAM-1 C-terminal structures. Received: 5 December 1996 / Revised: 2 January 1997     

Regulation of adipocyte differentiation and gene expression-crosstalk between TGFβ and wnt signaling pathways

Obesity results in reduced differentiation potential of adipocytes leading to adipose tissue insulin resistance. Elevated proinflammatory cytokines from adipose tissue in obesity, such as TNFα have been implicated in the reduced adipocyte differentiation. Other mediators of reduced adipocyte differentiation include TGFβ and wnt proteins. Although some overlap exists in the signaling cascades of the wnt and TGFβ pathways it is unknown if TGFβ or wnt proteins reciprocally induce the expression of each other to maximize their biological effects in adipocytes. Therefore, we investigated the possible involvement of TGFβ signaling in wnt induced gene expression and vice versa in 3T3-L1 adipocyte. Effect of TGFβ and Wnt pathways on differentiation was studied in preadipocytes induced to differentiate in the presence of Wnt3a or TGFβ1 and their inhibitors (FZ8-CRD and SB431542, respectively). Regulation of intracellular signaling and gene expression was also studied in mature adipocytes. Our results show that both TGFβ1 and Wnt3a lead to increased accumulation of β-catenin, phosphorylation of AKT and p44/42 MAPK. However, differences were found in the pattern of gene expression induced by the two proteins suggesting that distinct, but complex, signaling pathways are activated by TGFβ and wnt proteins to independently regulate adipocyte function.     

SnoN/SkiL,a TGFβ signaling mediator

Effective treatment of cancer cells with chemotherapeutic drugs relies on their ability to induce cell death, making the discovery of their mechanisms of action crucial. Arsenic trioxide (As₂O₃), used in the treatment of promyelocytic leukemia (PML), triggers cell death in several solid tumor cell lines including ovarian carcinomas. While As₂O₃ is remarkably cytotoxic in human ovarian cancer cells, its mechanism of action is poorly understood. We recently investigated the effects of As₂O₃ on several transforming growth factor-β (TGFβ) signaling mediators to better understand its cell death mechanism. Indeed, dysregulated (TGFβ) signaling is typical of ovarian cancers. Based on our findings, we propose that As₂O₃ induces a Beclin 1-independent autophagic pathway in ovarian carcinoma cells by modulating SnoN/SkiL expression, implicating SnoN as a novel therapeutic target for ovarian cancers.     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Cutis laxa: Intersection of elastic fiber biogenesis,TGFβ signaling,the secretory pathway and metabolism

Cutis laxa (CL), a disease characterized by redundant and inelastic skin, displays extensive locus heterogeneity. Together with geroderma osteodysplasticum and arterial tortuosity syndrome, which show phenotypic overlap with CL, eleven CL-related genes have been identified to date, which encode proteins within 3 groups. Elastin, fibulin-4, fibulin-5 and latent transforming growth factor-β-binding protein 4 are secreted proteins which form elastic fibers and are involved in the sequestration and subsequent activation of transforming growth factor-β (TGFβ). Proteins within the second group, localized to the secretory pathway, perform transport and membrane trafficking functions necessary for the modification and secretion of elastic fiber components. Key proteins include a subunit of the vacuolar-type proton pump, which ensures the efficient secretion of tropoelastin, the precursor or elastin. A copper transporter is required for the activity of lysyl oxidases, which crosslink collagen and elastin. A Rab6-interacting goglin recruits kinesin motors to Golgi-vesicles facilitating the transport from the Golgi to the plasma membrane. The Rab and Ras interactor 2 regulates the activity of Rab5, a small guanosine triphosphatase essential for the endocytosis of various cell surface receptors, including integrins. Proteins of the third group related to CL perform metabolic functions within the mitochondria, inhibiting the accumulation of reactive oxygen species. Two of these proteins catalyze subsequent steps in the conversion of glutamate to proline. The third transports dehydroascorbate into mitochondria. Recent studies on CL-related proteins highlight the intricate connections among membrane trafficking, metabolism, extracellular matrix assembly, and TGFβ signaling.     

Activation of renal profibrotic TGFβ controlled signaling cascades by calcineurin and mTOR inhibitors

The calcineurin inhibitors (CNI) cyclosporine A (CsA) and tacrolimus represent potent immunosuppressive agents frequently used for solid organ transplantation and treatment of autoimmune disorders. Despite of their immense therapeutic benefits, residual fibrosis mainly in the kidney represents a common side effect of long-term therapy with CNI. Regardless of the immunosuppressive action, an increasing body of evidence implicates that a drug-induced increase in TGFβ and subsequent activation of TGFβ-initiated signaling pathways is closely associated with the development and progression of CNI-induced nephropathy. Mechanistically, an increase in reactive oxygen species (ROS) generation due to drug-induced changes in the intracellular redox homeostasis functions as an important trigger of the profibrotic signaling cascades activated under therapy with CNI. Although, inhibitors of the mechanistic target of rapamycin (mTOR) kinase have firmly been established as alternative compounds with a lower nephrotoxic potential, an activation of fibrogenic signaling cascades has been reported for these drugs as well. This review will comprehensively summarize recent advances in the understanding of profibrotic signaling events modulated by these widely used compounds with a specific focus put on mechanisms occurring independent of their respective immunosuppressive action. Herein, the impact of redox modulation, the activation of canonical TGFβ and non-Smad pathways and modulation of autophagy by both classes of immunosuppressive drugs will be highlighted and discussed in a broader perspective. The comprehensive knowledge of profibrotic signaling events specifically accompanying the immunomodulatory activity of these widely used drugs is needed for a reliable benefit-risk assessment under therapeutic regimens.     

The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.     

Mapping ERβ Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ERβ Interactor

Considerable effort by numerous laboratories has resulted in an improved understanding of estrogen and SERM action mediated by the two estrogen receptors, ERα and ERβ. However, many of the targets for ERβ in cell physiology remain elusive. Here, the C4-12/Flag.ERβ cell line which stably expressed Flag.ERβ is used to study ERβ genomic functions without ERα interference. Mapping ERβ binding sites in these cells reveals ERβ unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERβ target genes. Gene ontology analysis reveals that ERβ targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERβ binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERβ binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERβ binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERβ genomic functions in our cell model, we confirm the anti-proliferative role of ERβ and discover the novel cross talk of ERβ with EBF1 which has various implications in normal physiology.