Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.     

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway

Members of the transforming growth factor β (TGF-β) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-β signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-β receptor (TGF-βR) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-βR internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-β-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-βR complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-βR endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.     

The Smad pathway in transforming growth factor-β signaling

The transforming growth factor b (TGF-b) superfamily comprises a great number of structurally related polypeptide growth factors, such as TGF-bs, activins, inhibins, bone morphogenic proteins (BMPs), growth differentiation factors (GDFs), M黮lerian inhibitory substance, and glial cell-derived neurotrophic factor (GDNF), etc[1]. The TGF-b superfamily members are multifunctional agonists involved in a broad spectrum of biological processes such as cell proliferation and differentiation, e…     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis

Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy or apoptosis. The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between NRF2/KEAP1 signaling and regulation of apoptosis and autophagy is not well understood. In this hypothesis article we discuss how KEAP1 protein and its direct interactants (such as PGAM5, prothymosin α, FAC1 (BPTF), and p62) provide a molecular foundation for a possible cross-talk between NRF2/KEAP1, apoptosis, and autophagy pathways. We present a hypothesis for how NRF2/KEAP1 may interfere with the cellular apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner-PGAM5. Based on very recent experimental evidence, new hypotheses for a cross-talk between NF-κB and the NRF2/KEAP1 pathway in the context of autophagy-related "molecular hub" protein p62 are also presented. The roles of KEAP1 molecular binding partners in apoptosis regulation during carcinogenesis and in neurodegenerative diseases are also discussed.     

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.     

Reciprocal cross-talk between Nod2 and TAK1 signaling pathways

Mutations in the leucine-rich repeat (LRR) domain of Nod2 have been implicated in the pathogenesis of Crohn's disease, yet the function of Nod2 and regulation of the Nod2 pathway remain unclear. In this study, we determined that mitogen-activated protein kinase kinase transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) interacts with Nod2 and is required for Nod2-mediated NF-kappaB activation. The dominant negative form of TAK1 abolished muramyl dipeptide-induced NF-kappaB activation in Nod2-expressing cells. Nod2, acting in a reciprocal manner, inhibited TAK1-induced NF-kappaB activation in RICK-deficient embryonic fibroblasts. Nod2 appears to interact with TAK1 through its LRR region to exert its inhibitory effect on TAK1-induced NF-kappaB activation. Further, wild-type LRR more effectively suppressed NF-kappaB activation induced by TAK1 than LRR with a 3020insC mutation. Considered together, these findings demonstrate a critical role for TAK1 in Nod2-mediated innate immune responses and reveal a novel function for Nod2 in the regulation of the TAK1 signaling pathway.     

Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways

Smad7 has been identified as a negative regulator of transforming growth factor beta (TGF-beta) signaling by interfering with the phosphorylation of other Smad proteins by TGF-beta receptor type I (TbetaRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF-beta stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF-beta signaling pathways. Consistent with previous studies, Smad7 inhibited TGF-beta-stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-beta-induced growth inhibition.     

Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta

Introduction  

Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.     

A genetic link between Tbx1 and fibroblast growth factor signaling

Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, as the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. We hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. We show that the expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1(-/-) mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, we crossed Tbx1 and Fgf8 mutants. Double heterozygous Tbx1(+/-);Fgf8(+/-) mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1(+/-);Fgf8(+/+) mutants, while Tbx1(+/+);Fgf8(+/-) animals are normal. We found that Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. Our data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. We speculate that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1.