Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

USP44 suppresses proliferation and enhances apoptosis in colorectal cancer cells by inactivating the Wnt/β‐catenin pathway via Axin1 deubiquitination

Colorectal cancer (CRC) is the leading cause of cancer death, and its 5‐year survival rate remains unsatisfactory. Recent studies have revealed that ubiquitin‐specific protease 44 (USP44) is a cancer suppressor or oncogene depending on the type of neoplasm. However, its role in CRC remains unclear. Here, we found that the USP44 expression level was markedly decreased in CRC, and USP44 overexpression inhibited proliferation while enhancing apoptosis in CRC cells, suggesting that USP44 is a cancer suppressor in CRC. We then investigated if USP44 functioned through regulating the Wnt/β‐catenin pathway. We found that USP44 overexpression increased the Axin1 protein while decreasing β‐catenin, c‐myc, and cyclin D1 proteins, suggesting that USP44 inhibited the activation of the Wnt/β‐catenin pathway. Moreover, we found that two Wnt/β‐catenin activators, LiCl and SKL2001, both attenuated oeUSP44‐mediated proliferation and apoptosis in CRC cells. Collectively, these data points indicated that USP44 inhibited proliferation while promoting apoptosis in CRC cells by inhibiting the Wnt/β‐catenin pathway. Interestingly, we observed that USP44 overexpression did not affect the Axin1 mRNA level. Further study uncovered that USP44 interacted with Axin1 and reduced the ubiquitination of Axin1. Furthermore, Axin1 knock‐down abolished the effects of oeUSP44 on proliferation, apoptosis, and Wnt/β‐catenin activity in CRC cells. Taken together, this study demonstrates that USP44 inhibits proliferation while enhancing apoptosis in CRC cells by inactivating the Wnt/β‐catenin pathway via Axin1 deubiquitination. USP44 is a cancer suppressor in CRC and a potential target for CRC therapy.     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

Due to an increasing emergence of new and drug‐resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β‐catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β‐catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up‐regulated and 20 miRNAs that down‐regulated the Wnt/β‐catenin signalling pathway. Fifteen miRNAs were validated to up‐regulate and five miRNAs to down‐regulate the pathway. Overexpression of four selected miRNAs (miR‐193b, miR‐548f‐1, miR‐1‐1, and miR‐509‐1) that down‐regulated the Wnt/β‐catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34‐infected HEK293 cells, and progeny virus production. Overexpression of miR‐193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR‐193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β‐catenin was a target of miR‐193b, and β‐catenin rescued miR‐193b‐mediated suppression of IAV infection. miR‐193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus‐mediated gene transfer of miR‐193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR‐193b represses IAV infection by inhibiting Wnt/β‐catenin signalling.     

TIPE1 suppresses invasion and migration through down‐regulating Wnt/β‐catenin pathway in gastric cancer

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.     

Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β‐catenin activity

Salidroside is a major phenylethanoid glycoside in Rhodiola rosea L., a traditional Chinese medicine, with multiple biological activities. It has been shown that salidroside possesses protective effects for alleviating diabetic renal dysfunction, contrast‐induced‐nephropathy and other kidney diseases. However, the involved molecular mechanism was still not understood well. Herein, we examined the protective effects of salidroside in mice with Adriamycin (ADR)‐induced nephropathy and the underlying molecular mechanism. The results showed that salidroside treatment ameliorates proteinuria; improves expressions of nephrin and podocin; and reduces kidney fibrosis and glomerulosclerosis induced by ADR. Mechanistically, ADR induces a robust accumulation of β‐catenin in the nucleus and stimulates its downstream target gene expression. The application of salidroside largely abolishes the nuclear translocation of β‐catenin and thus inhibits its activity. Furthermore, the activation of β‐catenin almost completely counteracts the protective roles of salidroside in ADR‐injured podocytes. Taken together, our data indicate that salidroside ameliorates proteinuria, renal fibrosis and podocyte injury in ADR nephropathy, which may rely on inhibition of β‐catenin signalling pathway.     

Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

Renal fibrosis is the common pathological feature in a variety of chronic kidney diseases. Aging is highly associated with the progression of renal fibrosis. Among several determinants, mitochondrial dysfunction plays an important role in aging. However, the underlying mechanisms of mitochondrial dysfunction in age‐related renal fibrosis are not elucidated. Herein, we found that Wnt/β‐catenin signaling and renin–angiotensin system (RAS) activity were upregulated in aging kidneys. Concomitantly, mitochondrial mass and functions were impaired with aging. Ectopic expression of Klotho, an antagonist of endogenous Wnt/β‐catenin activity, abolished renal fibrosis in d ‐galactose (d ‐gal)‐induced accelerated aging mouse model and significantly protected renal mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species. In an established aging mouse model, dickkopf 1, a more specific Wnt inhibitor, and the mitochondria‐targeted antioxidant mitoquinone restored mitochondrial mass and attenuated tubular senescence and renal fibrosis. In a human proximal tubular cell line (HKC‐8), ectopic expression of Wnt1 decreased biogenesis and induced dysfunction of mitochondria, and triggered cellular senescence. Moreover, d ‐gal triggered the transduction of Wnt/β‐catenin signaling, which further activated angiotensin type 1 receptor (AT1), and then decreased the mitochondrial mass and increased cellular senescence in HKC‐8 cells and primary cultured renal tubular cells. These effects were inhibited by AT1 blocker of losartan. These results suggest inhibition of Wnt/β‐catenin signaling and the RAS could slow the onset of age‐related mitochondrial dysfunction and renal fibrosis. Taken together, our results indicate that Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction.     

FDPS promotes glioma growth and macrophage recruitment by regulating CCL20 via Wnt/β‐catenin signalling pathway

Glioma is one of the most lethal tumours and common malignant in the central nervous system (CNS), which exhibits diffuse invasion and aggressive growth. Several studies have reported the association of FDPS to tumour development and progression. However, the role of FDPS in progression of glioma and macrophage recruitment is not well‐elucidated. In the current study, a remarkable enhancement in FDPS level was observed in glioma tissues and associated with poor prognosis, contributed to tumour growth. FDPS was correlated with macrophage infiltration in glioma and pharmacological deletion of macrophages largely abrogated the oncogenic functions of FDPS in glioma. Mechanistically, FDPS activated Wnt/β‐catenin signalling pathway and ultimately facilitates macrophage infiltration by inducing CCL20 expression. In conclusion, overexpressed FDPS exhibits an immunomodulatory role in glioma. Therefore, targeting FDPS may be an effective therapeutic strategy for glioma.     

MicroRNA‐130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/β‐catenin pathway

More and more studies indicate the relevance of miRNAs in inducing certain drug resistance. Our study aimed to investigate whether microRNA‐130b‐3p (miR‐130b) mediates the chemoresistance as well as proliferation of lung cancer (LC) cells. MTS assay and apoptosis analysis were conducted to determine cell proliferation and apoptosis, respectively. Binding sites were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT‐PCR and immunoblot, respectively. Mouse xenograft model was used to evaluate the role of miR‐130b in cisplatin resistance in vivo. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR ) versus its parental cell lines, indicated its crucial relevance for LC biology. We identified PTEN as miR‐130b's major target and inversely correlated with miR‐130b expression in LC. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. Suppression of miR‐130b enhanced drug cytotoxicity and reduced proliferation of A549/CR cells both internally and externally. Particularly, miR‐130b mediated Wnt/β‐catenin signalling pathway activities, chemoresistance and proliferation in LC cell, which was partially blocked following knockdown of PTEN. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway.