TGF-β/TGF-β receptor system and its role in physiological and pathological conditions

The TGF-β (transforming growth factor-β) system signals via protein kinase receptors and Smad mediators to regulate a plethora of biological processes, including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing and inflammation. In addition, alterations of specific components of the TGF-β signalling pathway may contribute to a broad range of pathologies such as cancer, cardiovascular pathology, fibrosis and congenital diseases. The knowledge about the mechanisms involved in TGF-β signal transduction has allowed a better understanding of the disease pathogenicity as well as the identification of several molecular targets with great potential in therapeutic interventions.     

Cardiomyocyte PDGFR-β signaling is an essential component of the mouse cardiac response to load-induced stress

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-β expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-β signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-β was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-β is a regulator of the compensatory cardiac response to pressure overload–induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.     

Pivotal role of leptin-hypothalamus signaling in the etiology of diabetes uncovered by gene therapy: a new therapeutic intervention?

The incidence of diabetes mellitus has soared to epidemic proportion worldwide. The debilitating chronic hyperglycemia is caused by either lack of insulin as in diabetes type 1 or its ineffectiveness as in diabetes type 2. Frequent replacement of insulin with or without insulin analogs for optimum glycemic control are the conventional cumbersome therapies. Recent application of leptin gene transfer technology has uncovered the participation of adipocytes-derived leptin-dependent hypothalamic neural signaling in glucose homeostasis and demonstrated that a breakdown in this communication due to leptin insufficiency in the hypothalamus underlies the etiology of chronic hyperglycemia. Reinstatement of central leptin sufficiency by hyperleptinemia produced either by intravenous leptin infusion or a single systemic injection of recombinant adenovirus vector encoding leptin gene suppressed hyperglycemia and evoked euglycemia only transiently in rodent models of diabetes type 1. In contrast, stable restoration of leptin sufficiency, solely in the hypothalamus, with biologically active leptin transduced by an intracerebroventicular injection of recombinant adeno-associated virus vector encoding leptin gene (rAAV-lep) abolished hyperglycemia and imposed euglycemia through the extended duration of experiment by stimulating glucose disposal in the periphery in models of diabetes type 1. Further, similar hypothalamic leptin transgene expression abrogated chronic hyperglycemia and hyperinsulinemia, the predisposing risk factors of the age and environmentally acquired diabetes type 2, and instituted euglycemia by independently activating relays that stimulate glucose metabolism and repress hyperinsulinemia and improve insulin sensitivity in the periphery. Consequently, this durable antidiabetic efficacy of one time rAAV-lep neurotherapy offers a potential novel substitute for insulin therapy following preclinical trials in subhuman primates and humans.     

Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice

Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.     

Cardiac fibroblasts in pressure overload hypertrophy: the enemy within?

Cardiac fibroblasts have been long recognized as active participants in heart disease; however, their exact physiological and pathological roles remain elusive, mainly due to the lack of specific markers. In this issue of the JCI, Moore-Morris and colleagues used a fibroblast-specific collagen1a1-GFP reporter to demonstrate that fibroblast accumulation after aortic banding in murine hearts arises almost exclusively from proliferation of resident fibroblasts originating from both the epicardium and a previously unrecognized source, the endocardium. Further characterization of fibroblast origin and function in different types and stages of heart disease could lead to development of improved fibroblast-targeted cardiac therapies.     

GSK-3α directly regulates β-adrenergic signaling and the response of the heart to hemodynamic stress in mice

The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, α and β. Although GSK-3β has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3α in the mouse heart using gene targeting. Gsk3a^–/– mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired β-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3α appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of β-adrenergic responsiveness. In the absence of GSK-3α, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of β-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3.     

Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling

During bone resorption, abundant factors previously buried in the bone matrix are released into the bone marrow microenvironment, which results in recruitment and differentiation of bone marrow mesenchymal stem cells (MSCs) for subsequent bone formation, temporally and spatially coupling bone remodeling. Parathyroid hormone (PTH) orchestrates the signaling of many pathways that direct MSC fate. The spatiotemporal release and activation of matrix TGF-β during osteoclast bone resorption recruits MSCs to bone-resorptive sites. Dysregulation of TGF-β alters MSC fate, uncoupling bone remodeling and causing skeletal disorders. Modulation of TGF-β or PTH signaling may reestablish coupled bone remodeling and be a potential therapy.     

Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor β (PDGFRβ) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-β–dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRβ-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions.     

TGF-β in the pathogenesis and prevention of disease: a matter of aneurysmic proportions

TGF-β regulates many aspects of cellular performance relevant to tissue morphogenesis and homeostasis. Postnatal perturbation of TGF-β signaling contributes to the pathogenesis of many disease states, as recently exemplified through the study of Marfan syndrome (MFS), including aortic aneurysm and skeletal muscle myopathy. Heterogeneity in the regulation and consequences of TGF-β signaling, amplified in the context of disease, has engendered confusion and controversy regarding its utility as a therapeutic target. Three studies recently published in the JCI, including one in this issue, underscore the complexity of this subject. Heydemann and colleagues implicate dimorphic variation in latent TGF-β–binding protein 4 (LTBP4), a regulator of TGF-β bioavailability and activation, as a modifier of muscular dystrophy in γ-sarcoglycan–deficient mice. In contrast to experience with ascending aortic aneurysm in MFS, Wang and colleagues show that systemic abrogation of TGF-β signaling worsens (rather than attenuates) Ang II–induced abdominal aortic aneurysm progression in mice. Tieu and colleagues define alterations in the regulation of vascular inflammation in the pathogenesis of Ang II–induced aneurysm and dissection in mice, which may help shed some light on this apparent paradox. Historically, perturbation (largely enhancement) of TGF-β signaling has been strongly implicated in the pathogenesis of diverse disease states, prominently including the initiation and progression of cancer and tissue fibrosis. Despite decades of intensive study in these contexts, the net effects of TGF-β signaling in disease pathogenesis and, perhaps more importantly, the incorporation of its antagonism into therapeutic strategies, remain controversial. This is nicely illustrated by the so-called TGF-β cancer paradox. In brief, TGF-β plays a prominent role in the suppression of tumorigenesis through induction of cell-cycle arrest and apoptosis and maintenance of cellular differentiation, as evidenced by the frequent biallelic loss of genes encoding TGF-β receptors or intracellular mediators of signaling in multiple tumor types (for example, see ref. 1). Attenuation or loss of tumor responsiveness can induce upregulation of TGF-β ligand expression, resulting in excessive stimulation of the signaling-intact neighboring stroma (reviewed in ref. 2). Attributable consequences include impairment of tumor surveillance through inhibition of adaptive immunity, acceleration of tumor growth through enhancement of angiogenesis, and induction of tumor invasion and metastasis through stimulation of innate immunity (including mast cell, monocyte, and macrophage chemotaxis), compromising of endothelial boundaries, and promotion of epithelial- or endothelial-mesenchymal transition (EpMT [ref. 2] and EnMT [ref. 3], respectively; collectively EMT). Amplification of TGF-β signaling can occur due to enhanced TGF-β ligand expression (e.g., by recruited mast cells or macrophages) or activation (e.g., by MMPs or simple fibrosis-dependent enhancement of shear stress within the tumor microenvironment; ref. 4). Additional TGF-β–related paradoxes are evident. For example, TGF-β can either induce or suppress angiogenesis depending upon its concentration and repertoire of TGF-β receptors and accessory proteins (2).     

Implication of γδ T cells in the human immune response to cytomegalovirus

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.     

Does amplitude-modulated frequency have a role in the hypoalgesic response of interferential current on pressure pain sensitivity in healthy subjects? A randomised crossover study

Objective

To investigate the hypoalgesic effect of amplitude-modulated frequency during interferential current therapy using an experimentally induced mechanical pain model in normal subjects. This study examined pain pressure sensitivities achieved when the amplitude-modulated frequency parameter was present (100 Hz) and absent (0 Hz).

Design

Randomised controlled crossover trial with repeated measures.

Setting

University research laboratory.

Participants

Forty-six healthy volunteers (23 males, 23 females).

Interventions

Two interferential therapy protocols (with and without amplitude-modulated frequencies) were applied to the lumbar area on two different days.

Main outcome measures

Pressure pain thresholds over the lumbar area were measured before, during and after application of the interferential therapy protocols.

Results

A three-way analysis of variance with repeated measures failed to show any statistically significant difference between the two protocols in modifying pressure pain threshold values (mean difference 0.017 kg/cm², 95% confidence interval −0.384 to 0.350, P = 0.93). Statistically significant differences were identified (P < 0.001) between measurements, indicating a comparable decrease in pain sensitivity in both groups. However, the increase in pressure pain thresholds (0.76 kg/cm²) failed to reach a level of clinical importance.

Conclusions

The addition of an amplitude-modulated frequency parameter to interferential therapy did not influence mechanical pain sensitivity in healthy subjects. Amplitude-modulated frequency is therefore unlikely to have a physiological hypoalgesic effect.     

EGFR-targeted therapy in colorectal cancer: does immunohistochemistry deserve a role in predicting the response to cetuximab?

The EGF receptor (EGFR) has emerged as a rational target for anticancer therapy for the treatment of colorectal cancer (CRC). Positive immunohistochemistry (IHC) staining for EGFR is used as a criterion for patient selection; however, doubt has been cast on the utility of this method. Not only is the response to cetuximab, an anti-EGFR mAb, low in patients expressing EGFRs, but a similar response to cetuximab has also been described in patients who do not express EGFRs. This review aims to evaluate the possible cause of the lack of correlation between the efficacy of cetuximab and EGFR IHC staining in CRC, as well as any modifications in the IHC method necessary to optimize patient selection for cetuximab therapy. In our opinion, the heterogeneous expression of the receptor in the neoplastic population and the inability of the mAbs used to predict the response to cetuximab could be the major cause of the failure of IHC staining as a reliable tool for patient selection. The use of specific mAbs directed against the phosphorylated and mutant form (EGFRvIII) of the EGFR could reinstate IHC as a valid predictor of response to therapy.     

A role for alcohol dehydrogenase in the Drosophila immune response?

In a recent study Drosophila larvae were injected with bacterial lipopolysaccharide (LPS) suspended in 1% ethanol and differentially induced protein fractions were identified. The levels of several proteins, including alcohol dehydrogenase (ADH), increased in LPS-treated flies and were labelled as immune response proteins. However, because control larvae were not injected with ethanol alone the identified proteins could represent a response to ethanol. Here, we injected Drosophila larvae with combinations of ethanol and LPS. While ADH activity increased in larvae receiving 1% ethanol, it was not increased after LPS injection. These results suggest that ADH plays no role in the Drosophila immune response, and that other proteins identified in the previous study may instead mediate ethanol tolerance in flies and other organisms.