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.     

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.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Aging,osteoarthritis and transforming growth factor-β signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-β (TGF-β). The impaired TGF-β signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

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.     

Interleukin-1β signaling in osteoarthritis – chondrocytes in focus

Osteoarthritis (OA) can be regarded as a chronic, painful and degenerative disease that affects all tissues of a joint and one of the major endpoints being loss of articular cartilage. In most cases, OA is associated with a variable degree of synovial inflammation. A variety of different cell types including chondrocytes, synovial fibroblasts, adipocytes, osteoblasts and osteoclasts as well as stem and immune cells are involved in catabolic and inflammatory processes but also in attempts to counteract the cartilage loss. At the molecular level, these changes are regulated by a complex network of proteolytic enzymes, chemokines and cytokines (for review: [1]). Here, interleukin-1 signaling (IL-1) plays a central role and its effects on the different cell types involved in OA are discussed in this review with a special focus on the chondrocyte.     

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.     

TGFβ superfamily signaling in the neural crest lineage

The neural crest cell (NCC) lineage is often referred to as the fourth germ layer in embryos, as its wide range of migration and early colonization of multiple tissues and organ systems throughout the developing body is astounding. Many human birth defects are thought to have their origins within the NCC lineage. Exciting recent conditional mouse targeting and transgenic combinatorial suppression approaches have revealed that the Tgf-b superfamily is a key signaling pathway within the cardiac and cranial NCC subpopulations. Given the complexity of Tgf-b superfamily signaling and that multiple ligand and receptor combinations have already been shown to be expressed within the NCC subpopulations, and the difficulty in transgenically targeting entire signaling cascades, we review several up-to-date transgenic approaches that are revealing unexpected consequences.     

TGFβ superfamily signaling in the neural crest lineage

The neural crest cell (NCC) lineage is often referred to as the fourth germ layer in embryos, as its wide range of migration and early colonization of multiple tissues and organ systems throughout the developing body is astounding. Many human birth defects are thought to have their origins within the NCC lineage. Exciting recent conditional mouse targeting and transgenic combinatorial suppression approaches have revealed that the TGFβ superfamily is a key signaling pathway within the cardiac and cranial NCC subpopulations. Given the complexity of TGFβ superfamily signaling and that multiple ligand and receptor combinations have already been shown to be expressed within the NCC subpopulations, and the difficulty in transgenically targeting entire signaling cascades, we review several up-to-date transgenic approaches that are revealing unexpected consequences.Key words: TGFβ, neural crest, heart, cranial crest, mouse transgenics     

p73 is required for endothelial cell differentiation,migration and the formation of vascular networks regulating VEGF and TGFβ signaling

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.Vascular system formation is one of the earliest events during organogenesis.¹ The original vascular plexus is established by vasculogenesis, through differentiation and assembly of mesodermal precursors.² The angiogenesis process allows the formation of new blood vessels from the existing vasculature and is perturbed in many diseases, including cancer.³ Although efforts have been made to identify factors that control vascular development, the understanding of the molecular networks remains incomplete.The formation of new capillaries and the remodeling of preexisting blood vessels is linked by signal transduction pathways.⁴ The members of the p53 family (p53, p73 and p63) coordinate cell proliferation, migration and differentiation, and could act as regulators of vascular development. TP73 function in angiogenesis is quite controversial,^{5, 6, 7} and it has never been addressed using developmental models.TP73 has a dual nature that resides in the existence of TA and DNp73 variants. TAp73 is capable of transactivating p53 targets^{8, 9, 10} whereas DNp73 can act as p53 and TAp73 repressor.^{11, 12, 13} TP73 final outcome will depend upon the differential expression of the TA/DNp73 isoforms in each cellular context, as they can execute synergic, as well as antagonist, functions.TP73 role during development is emphasized by the p73-knockout mice (Trp73−/−, p73KO from now on) multiple growth defects.¹⁴ These mice, which lack all p73 isoforms, exhibit gastrointestinal and cranial hemorrhages,¹⁴ suggestive of vascular fragility. Furthermore, TAp73 directly regulates GATA-1,⁸ which is essential for endothelial and hematopoietic differentiation.^{15, 16} This compounded information led us to hypothesize that p73 could be implicated in the regulation of vasculogenesis/angiogenesis.Regulation of these processes involves a broad range of signaling molecules essential for vascular growth and stability,¹⁷ such as vascular endothelial growth factor (VEGF)¹⁸ and transforming growth factor-β (TGF-β).¹⁹ TGF-β operates as a rheostat that controls endothelial cell (EC) differentiation, having an inhibitory effect on EC migration and proliferation by the TGF-β/TGFRI (ALK5)/Smad2/3 pathway, while the TβRII–ALK5/ALK1 complex activates Smad1/5/8, ID1 expression and a pro-angiogenic state.^{20, 21, 22}Regulation of the TGF-β and VEGF pathways by p53 family members has been documented.^{23, 24} However, p73''s function in these pathways during development remains largely unexplored. In this work, we have used mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSCs) as models that recapitulate early vascular morphogenesis.^{25, 26, 27} ESC and iPSC form multi-cellular aggregates (embryoid bodies, EBs) that, under appropriate conditions, generate functional EC.²⁸ mESC and iPSC differentiation capacity into ECs has been fully addressed.^{29, 30} We have also performed retinal vascularization analysis to assess vascular processes in vivo.^{31, 32}We demonstrate that p73 deficiency perturbs density and stability of mouse retinal development by affecting VEGF and TGF-β signaling. Furthermore, p73 is necessary for the assembly of vascular structures under physiological conditions in mESC and iPSC. Moreover, DNp73 positively affects angiogenesis through regulation of the TGF-β pathway in human umbilical vein cells (HUVEC) and DNp73-overexpression results in enhanced angiogenic potential of B16-F10 melanoma cells.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

TGFβ signaling in Tribolium: vertebrate-like components in a beetle

The cytokines of the TGFβ superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFβ subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFβ-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinβ and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFβ receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFβ signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle—and of the bee Apis mellifera—lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFβ signaling components and that TGFβ signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.