Regulation of LRRK2 mRNA stability by ATIC and its substrate AICAR through ARE-mediated mRNA decay in Parkinson's disease.

Mutations in LRRK2 are the most common genetic causes of Parkinson's disease (PD). While the enzymatic activity of LRRK2 has been linked to PD, previous work has also provided support for an important role of elevated LRRK2 protein levels, independent of enzymatic activity, in PD pathogenesis. However, the mechanisms underlying the regulation of LRRK2 protein levels remain unclear. Here, we identify a role for the purine biosynthesis pathway enzyme ATIC in the regulation of LRRK2 levels and toxicity. AICAr, the precursor of ATIC substrate, regulates LRRK2 levels in a cell-type-specific manner in vitro and in mouse tissue. AICAr regulates LRRK2 levels through AUF1-mediated mRNA decay. Upon AICAr treatment, the RNA binding protein AUF1 is recruited to the AU-rich elements (ARE) of LRRK2 mRNA leading to the recruitment of the decapping enzyme complex DCP1/2 and decay of LRRK2 mRNA. AICAr suppresses LRRK2 expression and rescues LRRK2-induced dopaminergic neurodegeneration and neuroinflammation in PD Drosophila and mouse models. Together, this study provides insight into a novel regulatory mechanism of LRRK2 protein levels and function via LRRK2 mRNA decay that is distinct from LRRK2 enzymatic functions.

Myotubularin functions through actomyosin to interact with the Hippo pathway.

The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin-mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. Here, we identify the phosphoinositide-3-phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11-mediated vesicular trafficking. We reveal PI(3)P dynamics as a novel mode of upstream regulation of actomyosin and establish Rab11-mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. Our study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2-related peripheral nerve myelination and human disorders.

Headcase and Unkempt Regulate Tissue Growth and Cell Cycle Progression in Response to Nutrient Restriction.

Nutrient restriction (NR) decreases the incidence and growth of many types of tumors, yet the underlying mechanisms are not fully understood. In this study, we identified Headcase (Hdc) and Unkempt (Unk) as two NR-specific tumor suppressor proteins that form a complex to restrict cell cycle progression and tissue growth in response to NR in Drosophila. Loss of Hdc or Unk does not confer apparent growth advantage under normal nutrient conditions but leads to accelerated cell cycle progression and tissue overgrowth under NR. Hdc and Unk bind to the TORC1 component Raptor and preferentially regulate S6 phosphorylation in a TORC1-dependent manner. We further show that HECA and UNK, the human counterparts of Drosophila Hdc and Unk, respectively, have a conserved function in regulating S6 phosphorylation and tissue growth. The identification of Hdc and Unk as two NR-specific tumor suppressors provides insight into molecular mechanisms underlying the anti-tumorigenic effects of NR.

D-chiro-inositol effectively attenuates cholestasis in bile duct ligated rats by improving bile acid secretion and attenuating oxidative stress.

Cholestatic liver diseases are important causes of liver cirrhosis and liver transplantation, but few drugs are available for treatment. D-chiro-inositol (DCI), an isomer of inositol found in many Leguminosae plants and in animal viscera, is used clinically for the treatment of polycystic ovary syndrome (PCOS) and diabetes mellitus. In this study, we investigated whether DCI exerted an anti-cholestatic effect and its underlying mechanisms. A cholestatic rat model was established via bile duct ligation (BDL). After the surgery, the rats were given DCI (150 mg·kg-1·d-1) in drinking water for 2 weeks. Oral administration of DCI significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and attenuated bile duct proliferation, parenchymal necrosis and fibrosis in BDL rats. Furthermore, DCI treatment significantly increased the serum and bile levels of total bile acid (TBA), and decreased TBA levels in the liver. Moreover, DCI treatment significantly increased expression of the genes encoding bile acid transporters BSEP (Abcb11) and MRP2 (Abcc2) in liver tissues. DCI treatment also markedly decreased hepatic CD68 and NF-kappaB (NF-κB) levels, significantly decreased the serum and hepatic MDA levels, markedly increased superoxide dismutase activity in both serum and liver tissues. Using whole-genome oligonucleotide microarray, we revealed that DCI treatment altered the expression profiles of oxidation reduction-related genes in liver tissues. Collectively, DCI effectively attenuates BDL-induced hepatic bile acid accumulation and decreases the severity of injury and fibrosis by improving bile acid secretion, repressing inflammation and decreasing oxidative stress. The results suggest that DCI might be beneficial for patients with cholestatic disorders.

The anti-hepatic fibrosis effects of dihydrotanshinone I are mediated by disrupting the yes-associated protein and transcriptional enhancer factor D2 complex and stimulating autophagy.

BACKGROUND AND PURPOSE: Dihydrotanshinone I (DHI), a lipophilic component of traditional Chinese medicine Salvia miltiorrhiza Bunge, has various therapeutic effects. We investigated the anti-fibrotic effect of DHI and its underlying mechanisms in vitro and in vivo. EXPERIMENTAL APPROACH: Rats subjected to bile duct ligation (BDL) were treated with DHI (25 mg·kg-1 ·day-1 , i.p.) for 14 days. Serum biochemical and liver tissue morphological analyses were performed. The human hepatic stellate cell line LX-2 served as a liver fibrosis model in vitro. Liver fibrogenic genes, yes-associated protein (YAP) downstream genes and autophagy markers were examined using western blot and real-time PCR analyses. Similar analyses were done in rat primary hepatic stellate cells (pHSCs). Autophagy flux was assessed by immunofluorescence. KEY RESULTS: In BDL rats, DHI administration attenuated liver necrosis, bile duct proliferation and collagen accumulation and reduced the expression of genes associated with fibrogenesis, including Tgfb1, Mmp-2, Acta2 and Col1a1. DHI (1, 5, 10 µmol·L-1 ) time- and dose-dependently suppressed the protein level of COL1A1, TGFß1 and α-SMA in LX-2 cells and rat pHSCs. Furthermore, DHI blocked the nuclear translocation of YAP, which inhibited the YAP/TEAD2 interaction and its downstream fibrogenic genes, connective tissue growth factor, SOX4 and survivin. This stimulated autophagic flux and accelerated the degradation of liver collagen. CONCLUSIONS AND IMPLICATIONS: DHI exerts anti-fibrotic effects in BDL rats, LX-2 cells and rat pHSCs by inhibiting the YAP and TEAD2 complex and stimulating autophagy. These findings indicate that DHI may be a potential therapeutic for the treatment of liver fibrosis.

Protective effect of gastrodin on bile duct ligation-induced hepatic fibrosis in rats.

Gastrodin has been showed to possess many beneficial physiological functions, including protection against inflammation and oxidation and apoptosis. Studies showed inflammation and oxidation play important roles in producing liver damage and initiating hepatic fibrogenesis. However, it has not been reported whether gastrodin has a protective effect against hepatic fibrosis or not. This is first ever made attempts to test gastrodin against liver fibrosis in bile duct ligation (BDL) rats. The aim of the present study is to evaluate the effect of gastrodin on BDL-induced hepatic fibrosis in rats. BDL rats were divided into two groups, BDL alone group, and BDL-gastrodin group treated with gastrodin (5 mg/ml in drinking water). The effects of gastrodin on BDL-induced hepatic injury and fibrosis in rats were estimated by assessing serum, urine, bile and liver tissue biochemistry followed by liver histopathology (using hematoxylin & eosin and sirius red stain) and hydroxyproline content measurement. The results showed that gastrodin treatment significantly reduced collagen content, bile duct proliferation and parenchymal necrosis after BDL. The serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) decreased with gastrodin treatment by 15.1 and 23.6 percent respectively in comparison to BDL group did not receive gastrodin. Gastrodin also significantly increased the level of serum high density lipoprotein (HDL) by 62.5 percent and down-regulated the elevated urine total bilirubin (TBIL) by 56.5 percent, but had no effect on total bile acid (TBA) in serum, bile and liver tissues. The immunohistochemical assay showed gastrodin remarkably reduced the expressions of CD68 and NF-κB in BDL rats. Hepatic SOD levels, depressed by BDL, were also increased by gastrodin by 8.4 percent. In addition, the increases of hepatic MDA and NO levels in BDL rats were attenuated by gastrodin by 31.3 and 38.7 percent separately. Our results indicate that gastrodin significantly attenuated the severity of BDL-induced hepatic injury and fibrosis by attenuating oxidative stress and inflammation. Taken together, these findings suggest that gastrodin might be an effective antifibrotic drug in cholestatic liver disease.

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats.

AIM: To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. METHODS: Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. RESULTS: Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-ß1 and α-smooth muscle actin. CONCLUSION: Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease.

Involvement of Ras GTPase-activating protein SH3 domain-binding protein 1 in the epithelial-to-mesenchymal transition-induced metastasis of breast cancer cells via the Smad signaling pathway.

In situ models of epithelial-to-mesenchymal transition (EMT)-induced carcinoma develop into metastatic carcinoma, which is associated with drug resistance and disease recurrence in human breast cancer. Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), an essential Ras mediator, has been implicated in cancer development, including cell growth, motility, invasion and apoptosis. Here, we demonstrated that the upregulation of G3BP1 activates the EMT in breast cancer cells. Silencing Smads almost completely blocked this G3BP1-induced EMT, suggesting that this process depends on the Smad signaling pathway. We also found that G3BP1 interacted with the Smad complex. Based on these results, we proposed that G3BP1 might act as a novel co-factor of Smads by regulating their phosphorylation status. Moreover, knockdown of G3BP1 suppressed the mesenchymal phenotype of MDA-MB-231 cells in vitro and suppressed tumor growth and lung metastasis of 4T1 cells in vivo. Our findings identified a novel function of G3BP1 in the progression of breast cancer via activation of the EMT, indicating that G3BP1 might represent a potential therapeutic target for metastatic human breast cancer.