Cerebral small vessel disease-related protease HtrA1 processes latent TGF-β binding protein 1 and facilitates TGF-β signaling

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.Cerebral small vessel disease (SVD) accounts for roughly one fifth of all strokes worldwide and is recognized as a major cause of cognitive decline and dementia (1). Familial forms of SVD have successfully been used as model conditions for mechanistic studies on SVD (2–4). CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy), a recessive SVD, is characterized by juvenile and recurrent strokes, extensive brain white matter lesions, and premature dementia (5). The disease is caused by mutations in the HTRA1 (high temperature requirement protein A1) gene (6) encoding an evolutionarily conserved serine protease (7). CARASIL mutations typically result in loss of HtrA1 activity, suggesting impaired substrate processing as a disease mechanism (6, 8, 9).HtrA1 has been shown to degrade a number of substrates, most of which are situated in the extracellular compartment, thus suggesting the extracellular space as primary location of HtrA1 function. HtrA1-mediated proteolysis has been implicated in various disease processes such as neurodegeneration (10, 11), age-related macular degeneration (12, 13), carcinogenesis (14) and arthritis (15). Only recently, identification of HTRA1 as the CARASIL-causing gene has highlighted its role in the vascular system and in TGF-β signaling, a well-defined regulator of angiogenesis and vascular homeostasis (16, 17) known to be implicated in several vascular conditions including Marfan Syndrome, Loeys–Dietz Syndrome, and hereditary hemorrhagic telangiectasia (18).Initial studies on CARASIL reported increased levels of the TGF-β prodomain (also called latency-associated peptide, LAP) and of TGF-β target genes in the cerebral vasculature of affected patients. This finding led to the proposition that up-regulation of the TGF-β pathway drives CARASIL pathogenesis (6, 8). However, the initial results were mostly obtained using overexpressing cells and autopsy material from advanced cases. Also, there is some controversy as to how HtrA1 interferes with TGF-β signaling. Proposed mechanisms include HtrA1-mediated extracellular cleavage of mature TGF-β (19, 20), cleavage of TGF-β receptors (21), and intracellular degradation of LAP (8).To better define the mechanistic link between HtrA1 and TGF-β and to identify physiological HtrA1 substrates relevant for CARASIL we investigated the effects of HtrA1 deficiency on the TGF-β pathway in brain tissue and embryonic fibroblasts from HTRA1 knockout mice and in skin fibroblasts from a CARASIL patient. Unexpectedly, we observed a consistent reduction of TGF-β activity in both murine and human material suggesting a facilitating role of HtrA1 in TGF-β signaling. We further identified latent TGF-β binding protein-1 (LTBP-1), a matricellular factor with a major role in TGF-β bioactivation (16, 22) as a novel physiological HtrA1 substrate and report on the functional consequences of its processing. Our findings point to a down-regulation of the TGF-β pathway in CARASIL pathogenesis and suggest LTBP-1 as a key HtrA1 substrate.     

TGF-β mediates suppression of adipogenesis by estradiol through connective tissue growth factor induction

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.     

Requirements of transcription factor Smad-dependent and -independent TGF-β signaling to control discrete T-cell functions

TGF-β modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-β achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-β signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously activated and produced effector cytokines in vivo on deletion of both Smad2 and Smad3. In addition, TGF-β failed to suppress T helper differentiation efficiently and to promote induced Treg generation of non-Treg cells lacking both Smad2 and Smad3, suggesting that Smad-dependent signaling is obligatory to mediate TGF-β function in non-Treg cells. Unexpectedly, however, the development, homeostasis, and function of Treg cells remained intact in the absence of Smad2 and Smad3, suggesting that the Smad-independent pathway is important for Treg function. Indeed, Treg-specific deletion of TGF-β-activated kinase 1 led to failed Treg homeostasis and lethal immune disorder in mice. Therefore, Smad-dependent and -independent TGF-β signaling discretely controls non-Treg and Treg function to modulate immune tolerance and immune homeostasis.     

GFRα1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling

The ability of cancer cells to invade along nerves is associated with aggressive disease and diminished patient survival rates. Perineural invasion (PNI) may be mediated by nerve secretion of glial cell line-derived neurotrophic factor (GDNF) attracting cancer cell migration through activation of cell surface Ret proto-oncogene (RET) receptors. GDNF family receptor (GFR)α1 acts as coreceptor with RET, with both required for response to GDNF. We demonstrate that GFRα1 released by nerves enhances PNI, even in the absence of cancer cell GFRα1 expression. Cancer cell migration toward GDNF, RET phosphorylation, and MAPK pathway activity are increased with exposure to soluble GFRα1 in a dose-dependent fashion. Dorsal root ganglia (DRG) release soluble GFRα1, which potentiates RET activation and cancer cell migration. In vitro DRG coculture assays of PNI show diminished PNI with DRG from GFRα1^+/− mice compared with GFRα1^+/+ mice. An in vivo murine model of PNI demonstrates that cancer cells lacking GFRα1 maintain an ability to invade nerves and impair nerve function, whereas those lacking RET lose this ability. A tissue microarray of human pancreatic ductal adenocarcinomas demonstrates wide variance of cancer cell GFRα1 expression, suggesting an alternate source of GFRα1 in PNI. These findings collectively demonstrate that GFRα1 released by nerves enhances PNI through GDNF-RET signaling and that GFRα1 expression by cancer cells enhances but is not required for PNI. These results advance a mechanistic understanding of PNI and implicate the nerve itself as a key facilitator of this adverse cancer cell behavior.Perineural invasion (PNI) is a mode of cancer progression in which tumor cells invade in, around, or along nerves (1). PNI is widely recognized as a highly adverse prognostic factor associated with paralysis, pain, paresthesia, increased cancer recurrence, and diminished patient survival (2, 3). PNI is a relatively common event for some cancer types including pancreatic, head and neck, prostate, stomach, colon, biliary tract, and other cancers (2–6).The molecular mechanisms underlying PNI remain poorly understood. Recent theories have suggested that nerve microenvironment may release chemotactic factors that attract cancer cells (2, 3). Glial cell line-derived neurotrophic factor (GDNF) is secreted by neurons and nerve supporting cells and plays a critical role in nerve development and axonal guidance. GDNF has been previously shown to be able to induce cancer cell migration (7, 8). GDNF first binds to GDNF family receptor (GFR)α1, which is a glycosyl-phosphatidylinositol (GPI)-anchored protein (9). This complex then binds to and activates the transmembrane Ret proto-oncogene (RET) receptor, inducing phosphorylation of RET tyrosine residues and initiating signal transduction (10). GFRα1 and RET must therefore interact together for a response to GDNF to occur through this receptor mechanism (9, 10). GDNF may also signal through alternate receptors including neural cell adhesion molecule (NCAM) and syndecan-3 (11–13).Our group has demonstrated that nerve-secreted GDNF serves as a key chemoattractant for cancer cells in the process of PNI. Activated RET on the cancer cell triggers the MAPK pathway and induces cell migration toward nerves in both in vitro and in vivo models of PNI (8). The inhibition of GDNF or RET inhibits this process. Therefore, GDNF-RET activity appears to be a significant mechanism of chemotactic signaling that participates in PNI. In these models, multiple cancer cell lines exhibiting PNI in response to GDNF expressed both RET and GFRα1.GDNF-RET signaling plays a fundamental role in nerve development and organogenesis. Interestingly, it has been previously shown that cell surface RET may be activated by GFRα1 cellular expression (cis) or by its noncellular presence (trans) in either a soluble or immobilized state (14, 15). Soluble GFRα1 molecules may capture GDNF and then present it to cell surface RET receptors for signal activation. GFRα1 can be released from the surface of neuronal cells, Schwann cells, and explants of sciatic nerve (14). Trauma to a nerve may facilitate such a release of GFRα1. We reasoned that the process of PNI entails a traumatic event to the nerve that might also lead to a release of soluble GFRα1.These concepts led to our current hypothesis that soluble GFRα1 released from nerves may enhance cancer cell PNI through activation of RET and downstream signal transduction. Findings from this study may improve our understanding of the mechanisms underlying PNI and elucidate the cancer cell requirements necessary for PNI to occur.     

Endothelial expression of hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-β signaling

Chronic systemic hypertension causes cardiac pressure overload leading to increased myocardial O(2) consumption. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of O(2) homeostasis. Mouse embryos lacking expression of the O(2)-regulated HIF-1α subunit die at midgestation with severe cardiac malformations and vascular regression. Here we report that Hif1a(f/f);Tie2-Cre conditional knockout mice, which lack HIF-1α expression only in Tie2(+) lineage cells, develop normally, but when subjected to pressure overload induced by transaortic constriction (TAC), they manifest rapid cardiac decompensation, which is accompanied by excess cardiac fibrosis and myocardial hypertrophy, decreased myocardial capillary density, increased myocardial hypoxia and apoptosis, and increased TGF-β signaling through both canonical and noncanonical pathways that activate SMAD2/3 and ERK1/2, respectively, within endothelial cells of cardiac blood vessels. TAC also induces dilatation of the proximal aorta through enhanced TGF-β signaling in Hif1a(f/f);Tie2-Cre mice. Inhibition of TGF-β signaling by treatment with neutralizing antibody or pharmacologic inhibition of MEK-ERK signaling prevented TAC-induced contractile dysfunction and pathological remodeling. Thus, HIF-1 plays a critical protective role in the adaptation of the heart and aorta to pressure overload by negatively regulating TGF-β signaling in endothelial cells. Treatment of wild-type mice with digoxin, which inhibits HIF-1α synthesis, resulted in rapid cardiac failure after TAC. Although digoxin has been used for decades as an inotropic agent to treat heart failure, it does not improve survival, suggesting that the countertherapeutic effects of digoxin observed in the TAC mouse model may have clinical relevance.     

Transforming growth factor (TGF)-β signaling in cardiac remodeling

Myocardial TGF-β expression is upregulated in experimental models of myocardial infarction and cardiac hypertrophy, and in patients with dilated or hypertrophic cardiomyopathy. Through its effects on cardiomyocytes, mesenchymal and immune cells, TGF-β plays an important role in the pathogenesis of cardiac remodeling and fibrosis. TGF-β overexpression in the mouse heart is associated with fibrosis and hypertrophy. Endogenous TGF-β plays an important role in the pathogenesis of cardiac fibrotic and hypertrophic remodeling, and modulates matrix metabolism in the pressure-overloaded heart. In the infarcted heart, TGF-β deactivates inflammatory macrophages, while promoting myofibroblast transdifferentiation and matrix synthesis through Smad3-dependent pathways. Thus, TGF-β may serve as the “master switchThis article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure”. for the transition of the infarct from the inflammatory phase to formation of the scar. Because of its crucial role in cardiac remodeling, the TGF-β system may be a promising therapeutic target for patients with heart failure. However, efforts to translate these concepts into therapeutic strategies, in order to prevent cardiac hypertrophy and fibrosis, are hampered by the complex, pleiotropic and diverse effects of TGF-β signaling, by concerns regarding deleterious actions of TGF-β inhibition and by the possibility of limited benefit in patients receiving optimal treatment with ACE inhibitors and β-adrenergic blockers. Dissection of the pathways responsible for specific TGF-β-mediated actions and understanding of cell-specific actions of TGF-β are needed to design optimal therapeutic strategies. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure”.     

A novel therapeutic approach to treating obesity through modulation of TGFβ signaling

Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.     

Connection between inflammation and carcinogenesis in gastrointestinal tract: Focus on TGF-β signaling

Inflammation is a primary defense process against various extracellular stimuli,such as viruses,pathogens,foods,and environmental pollutants.When cells respond to stimuli for short periods of time,it results in acute or physiological inflammation.However,if the stimulation is sustained for longer time or a pathological state occurs,it is known as chronic or pathological inflammation.Several studies have shown that tumorigenesis in the gastrointestinal (GI) tract is closely associated with chronic inflammati...