Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Targeting tumor O-glycosylation modulates cancer-immune-cell crosstalk and enhances anti-PD-1 immunotherapy in head and neck cancer.

Cells in the tumor microenvironment (TME) communicate via membrane-bound and secreted proteins, which are mostly glycosylated. Altered glycomes of malignant tumors influence behaviors of stromal cells. In this study, we showed that the loss of core-1 ß1,3-galactosyltransferase (C1GALT1)-mediated O-glycosylation suppressed tumor growth in syngeneic head and neck cancer mouse models. O-glycan truncation in tumor cells promoted the M1 polarization of macrophages, enhanced T-cell-mediated cytotoxicity, and reduced interleukin-6 (IL-6) levels in the secretome. Proteasomal degradation of IL-6 was controlled by the O-glycan at threonine 166. Both IL-6/IL-6R blockade and O-glycan truncation in tumor cells induced similar pro-inflammatory phenotypes in macrophages and cytotoxic T lymphocytes (CTLs). The combination of the O-glycosylation inhibitor itraconazole and anti-programmed cell death protein 1 (anti-PD-1) antibody effectively suppressed tumor growth in vivo. Collectively, our findings demonstrate that O-glycosylation in tumor cells governs their crosstalk with macrophages and CTLs. Thus, targeting O-glycosylation successfully reshapes the TME and consequently enhances the efficacy of anti-PD-1 therapy.

TMTC1 promotes invasiveness of ovarian cancer cells through integrins ß1 and ß4.

Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Although O-mannosyltransferase TMTC1 is highly expressed by ovarian cancer, its pathophysiological role in ovarian cancer remains unclear. Here, immunohistochemistry showed that TMTC1 was overexpressed in ovarian cancer tissues compared with adjacent normal ovarian tissues, and high TMTC1 expression was associated with poor prognosis in patients with ovarian cancer. Silencing TMTC1 reduced ovarian cancer cell viability, migration, and invasion in vitro, as well as suppressed peritoneal tumor growth and metastasis in vivo. Moreover, TMTC1 knockdown reduced cell-laminin adhesion, which was associated with the decreased phosphorylation of FAK at pY397. Conversely, TMTC1 overexpression promoted these malignant properties in ovarian cancer cells. Glycoproteomic analysis and Concanavalin A (ConA) pull-down assays showed that integrins ß1 and ß4 were novel O-mannosylated protein substrates of TMTC1. Furthermore, TMTC1-mediated cell migration and invasion were significantly reversed by siRNA-mediated knockdown of integrin ß1 or ß4. Collectively, these results suggest that TMTC1-mediated invasive behaviors are primarily through integrins ß1 and ß4 and that TMTC1 is a potential therapeutic target for ovarian cancer.

Truncation of GalNAc-type O-glycans Suppresses CD44-mediated Osteoclastogenesis and Bone Metastasis in Breast Cancer.

The glycoprotein CD44 is a key regulator of malignant behaviors in breast cancer cells. To date, hyaluronic acid (HA)-CD44 signaling pathway has been widely documented in the context of metastatic bone diseases. Core 1 ß1,3-galactosyltransferase (C1GALT1) is a critical enzyme responsible for the elongation of O-glycosylation. Aberrant O-glycans is recognized as a hallmark in cancers. However, the effects of C1GALT1 on CD44 signaling and bone metastasis remain unclear. In this study, IHC analysis indicated that C1GALT1 expression positively correlates with CD44 in breast cancer. Silencing C1GALT1 accumulates the Tn antigen on CD44, which decreases CD44 levels and osteoclastogenic signaling. Mutations in the O-glycosites on the stem region of CD44 impair its surface localization as well as suppress cell-HA adhesion and osteoclastogenic effects of breast cancer cells. Furthermore, in vivo experiments demonstrated the inhibitory effect of silencing C1GALT1 on breast cancer bone metastasis and bone loss. In conclusion, our study highlights the importance of O-glycans in promoting CD44-mediated tumorigenic signals and indicates a novel function of C1GALT1 in driving breast cancer bone metastasis. IMPLICATIONS: Truncation of GalNAc-type O-glycans by silencing C1GALT1 suppresses CD44-mediated osteoclastogenesis and bone metastasis in breast cancer. Targeting the O-glycans on CD44 may serve as a potential therapeutic target for blocking cancer bone metastasis.

Development of triamcinolone acetonide-hyaluronic acid conjugates with selective targeting and less osteoporosis effect for rheumatoid arthritis treatments.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease in developed countries. In clinical treatment, steroids have been used as bridging and adjunctive therapy after disease-modifying anti-rheumatic drug administration. However, the severe side effects caused by the nonspecific targeting of organs followed by long-term administration have limited their usage in RA. In this study, poorly water-soluble triamcinolone acetonide (TA), a highly potent corticosteroid for intra-articular injection, is conjugated on hyaluronic acid (HA) for intravenous purposes with increased specific drug accumulation in inflamed parts for RA. Our results demonstrate that the designed HA/TA coupling reaction reveals >98 % conjugation efficiency in the dimethyl sulfoxide/water system, and the resulting HA-TA conjugates show lower osteoblastic apoptosis compared with that in free TA-treated osteoblast-like NIH3T3 cells. Furthermore, in a collagen-antibody-induced arthritis animal study, HA-TA conjugates enhanced the initiative targeting ability to inflame tissue and reduce the histopathological arthritic changes (score = 0). Additionally, the level of bone formation marker P1NP in HA-TA-treated ovariectomized mice (303.6 ± 40.6 pg/mL) is significantly higher than that in the free TA-treated group (143.1 ± 3.9 pg/mL), indicating the potential for osteoporotic reduction using an efficient HA conjugation strategy for the long-term administration of steroids against RA.

GALNT2 promotes invasiveness of colorectal cancer cells partly through AXL.

GalNAc-type O-glycosylation and its initiating GalNAc transferases (GALNTs) play crucial roles in a wide range of cellular behaviors. Among 20 GALNT members, GALNT2 is consistently associated with poor survival of patients with colorectal cancer in public databases. However, its clinicopathological significance in colorectal cancer remains unclear. In this study, immunohistochemistry showed that GALNT2 was overexpressed in colorectal tumors compared with the adjacent nontumor tissues. GALNT2 overexpression was associated with poor survival of colorectal cancer patients. Forced expression of GALNT2 promoted migration and invasion as well as peritoneal metastasis of colorectal cancer cells. In contrast, GALNT2 knockdown with siRNAs or knockout with CRISPR/Cas9 system suppressed these malignant properties. Interestingly, we found that GALNT2 modified O-glycans on AXL and determined AXL levels via the proteasome-dependent pathway. In addition, the GALNT2-promoted invasiveness was significantly reversed by AXL siRNAs. These findings suggest that GALNT2 promotes colorectal cancer invasion at least partly through AXL.

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153.

Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked ß-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

C1GALT1 expression predicts a favorable prognosis and suppresses malignant phenotypes via TrkA signaling in neuroblastoma.

Neuroblastoma (NB) is a childhood tumor derived from the sympathoadrenal lineage of the neural crest progenitor cells. Core 1 ß1,3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation, and its altered expression affects cancer behaviors. However, the role of C1GALT1 in NB tumors remains unclear. Our data showed that C1GALT1 expression was significantly associated with differentiated tumor histology, correlated with TrkA expression, and predicted good prognosis independently in NB. Downregulation of C1GALT1 promotes malignant behaviors of NB cells in vitro and in vivo. Mechanistic investigation showed that knockdown of C1GALT1 in NB cells increased TrkA pulled down through Vicia villosa agglutinin beads, indicating the modulation of O-glycans on TrkA by C1GALT1, and silencing C1GALT1 suppressed the TrkA expression on the NB cell surface. Overexpression of C1GALT1 increased the protein levels of TrkA and promoted the differentiation of NB cells, whereas knockdown of TrkA inhibited C1GALT1-induced neuronal differentiation. Moreover, the inhibitory effects of migration and invasion in C1GALT1-overexpressing NB cells were blocked by TrkA downregulation. C1GALT1 knockdown enhanced AKT phosphorylation but attenuated ERK phosphorylation, and these properties were consistent in C1GALT1-overexpressing NB cells with TrkA knockdown. Taken together, our data provided the first evidence for the existence of GalNAc-type O-glycans on TrkA and altered O-glycan structures by C1GALT1 can regulate TrkA signaling in NB cells. This study sheds light on the novel prognostic role of C1GALT1 in NB and provides new information of C1GALT1 and TrkA on the pathogenesis of NB.

TGFß promotes fibrosis by MYST1-dependent epigenetic regulation of autophagy.

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-ß (TGFß) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFß induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFß on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFß-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFß signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.

Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells.

Inflammatory diseases such as arthritis are chronic conditions that fail to resolve spontaneously. While the cytokine and cellular pathways triggering arthritis are well defined, those responsible for the resolution of inflammation are incompletely characterized. Here we identified interleukin (IL)-9-producing type 2 innate lymphoid cells (ILC2s) as the mediators of a molecular and cellular pathway that orchestrates the resolution of chronic inflammation. In mice, the absence of IL-9 impaired ILC2 proliferation and activation of regulatory T (Treg) cells, and resulted in chronic arthritis with excessive cartilage destruction and bone loss. In contrast, treatment with IL-9 promoted ILC2-dependent Treg activation and effectively induced resolution of inflammation and protection of bone. Patients with rheumatoid arthritis in remission exhibited high numbers of IL-9+ ILC2s in joints and the circulation. Hence, fostering IL-9-mediated ILC2 activation may offer a novel therapeutic approach inducing resolution of inflammation rather than suppression of inflammatory responses.

Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis.

OBJECTIVES: Notch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice. METHODS: Notch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments. RESULTS: Notch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1. CONCLUSIONS: Inhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA.

Inactivation of autophagy ameliorates glucocorticoid-induced and ovariectomy-induced bone loss.

OBJECTIVES: Autophagy has recently been shown to regulate osteoclast activity and osteoclast differentiation. Here, we aim to investigate the impact of autophagy inhibition as a potential therapeutic approach for the treatment of osteoporosis in preclinical models. METHODS: Systemic bone loss was induced in mice by glucocorticoids and by ovariectomy (OVX). Autophagy was targeted by conditional inactivation of autophagy-related gene 7 (Atg7) and by treatment with chloroquine (CQ). Bone density was evaluated by microCT. The role of autophagy on osteoclastogenesis was analysed by osteoclastogenesis and bone resorption assays. The quantification of receptor activator of nuclear factor κ B ligand and osteoprotegerin proteins in cocultures was performed using ELISA whereas that of osteoclast and osteoblast differentiation markers was by qPCR. RESULTS: Selective deletion of Atg7 in monocytes from Atg7(fl/fl)_x_LysM-Cre mice mitigated glucocorticoid-induced and OVX-induced osteoclast differentiation and bone loss compared with Atg7(fl/fl) littermates. Pharmacological inhibition of autophagy by treatment with CQ suppressed glucocorticoid-induced osteoclastogenesis and protected mice from bone loss. Similarly, inactivation of autophagy shielded mice from OVX-induced bone loss. Inhibition of autophagy led to decreased osteoclast differentiation with lower expression of osteoclast markers such as NFATc1, tartrate-resistant acid phosphatase, OSCAR and cathepsin K and attenuated bone resorption in vitro. In contrast, osteoblast differentiation was not affected by inhibition of autophagy. CONCLUSIONS: Pharmacological or genetic inactivation of autophagy ameliorated glucocorticoid-induced and OVX-induced bone loss by inhibiting osteoclastogenesis. These findings may have direct translational implications for the treatment of osteoporosis, since inhibitors of autophagy such as CQ are already in clinical use.

Inhibition of casein kinase II reduces TGFß induced fibroblast activation and ameliorates experimental fibrosis.

OBJECTIVES: Casein kinase II (CK2) is a constitutively active serine/threonine protein kinase that plays a key role in cellular transformation and tumorigenesis. The purpose of the study was to characterise whether CK2 contributes to the pathologic activation of fibroblasts in patients with SSc and to evaluate the antifibrotic potential of CK2 inhibition. METHODS: Activation of CK2, JAK2 and STAT3 in human skin and in experimental fibrosis was analysed by immunohistochemistry. CK2 signalling was inhibited by the selective CK2 inhibitor 4, 5, 6, 7-Tetrabromobenzotriazole (TBB). The mouse models of bleomycin-induced and TGFß receptor I (TBR)-induced dermal fibrosis were used to evaluate the antifibrotic potential of specific CK2 inhibition in vivo. RESULT: Increased expression of CK2 was detected in skin fibroblasts of SSc patients. Inhibition of CK2 by TBB abrogated the TGFß-induced activation of JAK2/STAT3 signalling and prevented the stimulatory effects of TGFß on collagen release and myofibroblasts differentiation in cultured fibroblasts. Inhibition of CK2 prevented bleomycin-induced and TBR-induced skin fibrosis with decreased dermal thickening, lower myofibroblast counts and reduced accumulation of collagen. Treatment with TBB also induced regression of pre-established fibrosis. The antifibrotic effects of TBB were accompanied by reduced activation of JAK2/STAT3 signalling in vivo. CONCLUSIONS: We provide evidence that CK2 is activated in SSc and contributes to fibroblast activation by regulating JAK2/STAT3 signalling. Inhibition of CK2 reduced the pro-fibrotic effects of TGFß and inhibited experimental fibrosis. Targeting of CK2 may thus be a novel therapeutic approach for SSc and other fibrotic diseases.

Signature of circulating microRNAs in osteoarthritis.

BACKGROUND: Osteoarthritis is the most common form of arthritis and a major socioeconomic burden. Our study is the first to explore the association between serum microRNA levels and the development of severe osteoarthritis of the knee and hip joint in the general population. METHODS: We followed 816 Caucasian individuals from 1995 to 2010 and assessed joint arthroplasty as a definitive outcome of severe osteoarthritis of the knee and hip. After a microarray screen, we validated 12 microRNAs by real-time PCR in the entire cohort at baseline. RESULTS: In Cox regression analysis, three microRNAs were associated with severe knee and hip osteoarthritis. let-7e was a negative predictor for total joint arthroplasty with an adjusted HR of 0.75 (95% CI 0.58 to 0.96; p=0.021) when normalised to U6, and 0.76 (95% CI 0.6 to 0.97; p=0.026) after normalisation to the Ct average. miRNA-454 was inversely correlated with severe knee or hip osteoarthritis with an adjusted HR of 0.77 (95% CI 0.61 to 0.97; p=0.028) when normalised to U6. This correlation was lost when data were normalised to Ct average (p=0.118). Finally, miRNA-885-5p showed a trend towards a positive relationship with arthroplasty when normalised to U6 (HR 1.24; 95% CI 0.95 to 1.62; p=0.107) or to Ct average (HR 1.30; 95% CI 0.99 to 1.70; p=0.056). CONCLUSIONS: Our study is the first to identify differentially expressed circulating microRNAs in osteoarthritis patients necessitating arthroplasty in a large, population-based cohort. Among these microRNAs, let-7e emerged as potential predictor for severe knee or hip osteoarthritis.

Combined inhibition of morphogen pathways demonstrates additive antifibrotic effects and improved tolerability.

OBJECTIVES: The morphogen pathways Hedgehog, Wnt and Notch are attractive targets for antifibrotic therapies in systemic sclerosis. Interference with stem cell regeneration, however, may complicate the use of morphogen pathway inhibitors. We therefore tested the hypothesis that combination therapies with low doses of Hedgehog, Wnt and Notch inhibitors maybe safe and effective for the treatment of fibrosis. METHODS: Skin fibrosis was induced by bleomycin and by overexpression of a constitutively active TGF-ß receptor type I. Adverse events were assessed by clinical monitoring, pathological evaluation and quantification of Lgr5-positive intestinal stem cells. RESULTS: Inhibition of Hedgehog, Wnt and Notch signalling dose-dependently ameliorated bleomycin-induced and active TGF-ß receptor type I-induced fibrosis. Combination therapies with low doses of Hedgehog/Wnt inhibitors or Hedgehog/Notch inhibitors demonstrated additive antifibrotic effects in preventive as well as in therapeutic regimes. Combination therapies were well tolerated. In contrast with high dose monotherapies, combination therapies did not reduce the number of Lgr5 positive intestinal stem cells. CONCLUSIONS: Combined inhibition of morphogen pathways exerts additive antifibrotic effects. Combination therapies are well tolerated and, in contrast to high dose monotherapies, may not impair stem cell renewal. Combined targeting of morphogen pathways may thus help to overcome dose-limiting toxicity of Hedgehog, Wnt and Notch signalling.

The Wnt antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation in systemic sclerosis.

OBJECTIVES: Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. METHODS: The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2'-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for ß-catenin. RESULTS: The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated experimental fibrosis. CONCLUSIONS: We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.

Inactivation of evenness interrupted (EVI) reduces experimental fibrosis by combined inhibition of canonical and non-canonical Wnt signalling.

OBJECTIVES: Canonical as well as non-canonical Wnt signalling pathways have emerged as core pathways of fibrosis. Their profibrotic effects are mediated via distinct intracellular cascades independently of each other. Thus, inhibition of both pathways may have additive antifibrotic effects. Here, we knocked down evenness interrupted (EVI) to simultaneously target for the first time canonical and non-canonical Wnt signalling in experimental fibrosis. METHODS: The antifibrotic effects of siRNA-mediated knockdown of EVI were evaluated in the mouse models of bleomycin-induced skin fibrosis and in fibrosis induced by adenoviral overexpression of a constitutively active TGF-ß receptor I (AdTBRI). RESULTS: Knockdown of EVI decreased the release of canonical and non-canonical Wnt ligands by fibroblasts and reduced the activation of canonical and non-canonical Wnt cascades in experimental fibrosis with decreased accumulation of ß-catenin and phosphorylated JNK and cJun. Inactivation of EVI exerted potent antifibrotic effects and reduced dermal thickening, myofibroblast differentiation and accumulation of collagen in the mouse models of bleomycin-induced and AdTBR-induced fibrosis. CONCLUSIONS: Inhibition of Wnt secretion by knockdown of EVI inhibits canonical and non-canonical Wnt signalling and effectively reduces experimental fibrosis in different preclinical models. Inhibition of Wnt secretion may thus be an interesting approach for the treatment of fibrosis.

The molecular chaperone Cosmc enhances malignant behaviors of colon cancer cells via activation of Akt and ERK.

Expression of T antigen (Galbeta1, 3GalNAc) is associated with enhanced metastatic potential and poor prognosis in colorectal cancer. Cosmc is a molecular chaperone required for the formation of an active T-synthase, which catalyzes the synthesis of T antigen. However, the expression and role of Cosmc in colorectal cancer are still unclear. Here, real-time PCR showed that overexpression of Cosmc mRNA in colorectal tumors compared with paired non-tumorous tissues was associated with increased American Joint Committee on Cancer (AJCC) tumor stage. Forced expression of Cosmc in HCT116 cells significantly increased T antigen expression and enhanced cell growth, migration, and invasion, which was associated with increased phosphorylation of focal adhesion kinase (FAK), ERK, and Akt. These Cosmc-enhanced malignant phenotypes were significantly suppressed by specific inhibitor of MEK or PI3K. We also found that Cosmc overexpression increased tumor growth and decreased survival of tumor-bearing SCID mice. Conversely, knockdown of Cosmc with siRNA in SW480 cells decreased malignant behaviors and the signaling pathways, which were substantially reversed by constitutively active Akt or MEK. Taken together, these results suggest that Cosmc promotes malignant phenotypes of colon cancer cells mainly via activation of MEK/ERK and PI3K/Akt signaling pathways, and that Cosmc may serve as a potential target for colorectal cancer treatment.

Autophagy: a key pathway of TNF-induced inflammatory bone loss.

Autophagy describes the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. Autophagy is essentially required to prevent accumulation of cellular damage and to ensure cellular homeostasis. Indeed, impaired autophagy has been implicated in a variety of different diseases. We examined the role of autophagy in inflammatory bone loss. We demonstrated that autophagy is activated by the pro-inflammatory cytokine tumor necrosis factor (TNF/TNFα) in osteoclasts of patients with rheumatoid arthritis (RA). Autophagy induces osteoclast differentiation and stimulates osteoclast-mediated bone resorption in vitro and in vivo, thereby highlighting autophagy as a novel mediator of TNF-induced bone resorption.

Blockade of canonical Wnt signalling ameliorates experimental dermal fibrosis.

BACKGROUND AND OBJECTIVES: Fibrosis is a major socioeconomic burden, but effective antifibrotic therapies are not available in the clinical routine. There is growing evidence for a central role of Wnt signalling in fibrotic diseases such as systemic sclerosis, and we therefore evaluated the translational potential of pharmacological Wnt inhibition in experimental dermal fibrosis. METHODS: We examined the antifibrotic effects of PKF118-310 and ICG-001, two novel inhibitors of downstream canonical Wnt signalling, in the models of prevention and treatment of bleomycin-induced dermal fibrosis as well as in experimental dermal fibrosis induced by adenoviral overexpression of a constitutively active transforming growth factor (TGF)-ß receptor I. RESULTS: PKF118-310 and ICG-001 were well tolerated throughout all experiments. Both therapeutic approaches showed antifibrotic effects in preventing and reversing bleomycin-induced dermal fibrosis as measured by skin thickness, hydroxyproline content and myofibroblast counts. PKF118-310 and ICG-001 were effective in inhibiting TGF-ß receptor I-driven fibrosis as assessed by the same outcome measures. CONCLUSIONS: Blockade of canonical Wnt signalling by PKF118-310 and ICG-001 showed antifibrotic effects in different models of skin fibrosis. Both therapies were well tolerated. Although further experimental evidence for efficacy and tolerability is necessary, inhibition of canonical Wnt signalling is a promising treatment approach for fibrosis.