Groundwater vulnerability and contamination risk mapping of semi-arid Totko river basin, India using GIS-based DRASTIC model and AHP techniques.

Totko river basin is a semi-arid watershed, which undergoes severe water crisis during the dry season. Presently, due to increase in population, demand of food has increased leading to a rise in growth of high yield crop variety and usage of chemical fertilizers and pesticides. So, surface water as well as groundwater is getting polluted. In this study assessment of groundwater vulnerability of Totko river basin has been done using DRASTIC and Analytic Hierarchy Process (AHP) models. For this, seven hydrogeological parameters have been considered which are; Depth to water level (D), Net Recharge (R), Aquifer media (A), Soil media (S), Topography (T), Impact of vadose zone (I) and Hydraulic Conductivity (C). Weight and rating analysis of the seven criteria and their sub-criteria have been done using generic DRASTIC algorithm and AHP comparison matrix. Ground Water Vulnerability Map (GWV) obtained from DRASTIC and AHP analysis has been divided into five vulnerable classes. Area of very high vulnerable zone is 6.53% more in AHP based vulnerability as compared to Generic DRASTIC. Similarly, these regions show a high nitrate concentration (30-50 ppm) in groundwater. GWV maps have been validated through nitrate concentration and the accuracy of the models have been assessed through Pearson's correlation coefficient and Kappa coefficient. To prevent groundwater contamination proper land use planning and watershed management are necessary, for which vulnerable zones need to be demarcated and DRASTIC is a useful model for vulnerability assessment.

Assessment of household water consumption during COVID-19 pandemic: a cross-sectional web-based study in India.

The fatal novel COVID-19 creates precarious threats to humans through speedy diffusion. This virus not only disrupts human health but also makes multidirectional loss and slowdown of modern earth. Almost all countries strictly imposed lockdown and social distancing norms to prevent the infection of COVID-19 virus. In almost all parts of the world, people are using more water for washing, cleaning, bathing and hand washing practices. As a result, per capita water demand along with expenditure have been significantly increased. The principal objective of this study is to evaluate the household level water consumption status and to improve the water security with management for future. The current study has been conducted among the general population of India to assess household level water consumption through internet E-Surveys Google form from August 18 to September 8, 2020. Around 1850 respondents have sent their comments from different sites (rural and urban areas in various climatic regions) of Indian subcontinent. The results show the tremendous increase of water usage along with electrical consumption and expenditure during COVID-19 pandemic situation. Our results revels that 10%, 15% and 17% of higher water consumption per day in rural, urban and peri-urban residential respectively. We hypothesize that the reasons for the increasing water demand and household consumption per day may be found in changed behavioral routines through bathing, washing clothes, and hand washing practices. This web-based study also suggests that few alternative and dependable management techniques i.e. rain water harvesting can be installed to fight the crisis and for the sustainable future. Subsequently, research and development are highly required for long-term management of water resources or reuse of water.

Haploinsufficient tumor suppressor PRP4K is negatively regulated during epithelial-to-mesenchymal transition.

The pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro, and cancer metastasis in mice. These results are consistent with PRP4K being a haploinsufficient tumor suppressor. Increased cell migration and invasion is associated with epithelial-to-mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non-transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple-negative breast cancer cells (MDA-MB-231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA-MB-231 and MCF10A cells and without changes in E-cadherin. Induction of EMT in MCF10A cells, by treatment with WNT-5a and TGF-ß1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT-5a/TGF-ß1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumor suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

High glucose-stimulated enhancer of zeste homolog-2 (EZH2) forces suppression of deptor to cause glomerular mesangial cell pathology.

Function of mTORC1 and mTORC2 has emerged as a driver of mesangial cell pathologies in diabetic nephropathy. The mechanism of mTOR activation is poorly understood in this disease. Deptor is a constitutive subunit and a negative regulator of both mTOR complexes. Mechanistic investigation in mesangial cells revealed that high glucose decreased the expression of deptor concomitant with increased mTORC1 and mTORC2 activities, induction of hypertrophy and, expression of fibronectin and PAI-1. shRNAs against deptor mimicked these pathologic outcomes of high glucose. Conversely, overexpression of deptor significantly inhibited all effects of high glucose. To determine the mechanism of deptor suppression, we found that high glucose significantly increased the expression of EZH2, resulting in lysine-27 tri-methylation of histone H3 (H3K27Me3). Employing approaches including pharmacological inhibition, shRNA-mediated downregulation and overexpression of EZH2, we found that EZH2 regulates high glucose-induced deptor suppression along with activation of mTOR, mesangial cell hypertrophy and fibronectin/PAI-1 expression. Moreover, expression of hyperactive mTORC1 reversed shEZH2-mediated inhibition of hypertrophy and expression of fibronectin and PAI-1 by high glucose. Finally, in renal cortex of diabetic mice, we found that enhanced expression of EZH2 is associated with decreased deptor levels and increased mTOR activity and, expression of fibronectin and PAI-1. Together, our findings provide a novel mechanism for mTOR activation via EZH2 to induce mesangial cell hypertrophy and matrix expansion during early progression of diabetic nephropathy. These results suggest a strategy for leveraging the intrinsic effect of deptor to suppress mTOR activity via reducing EZH2 as a novel therapy for diabetic nephropathy.

Groundwater vulnerability assessment using GIS-based DRASTIC model in Nangasai River Basin, India with special emphasis on agricultural contamination.

Nangasai basin is a semi-arid watershed where agriculture is the main source of economy. In present day, increasing population demands increase in food productivity which leads to increase use of fertilizers and chemical pesticides in agriculture. These fertilizers on the other hand mix up with the groundwater and increase the pollution, which affects human health adversely. So, for controlling the groundwater contamination risk proper water resource management and assessment of groundwater vulnerability is extremely important. Total 7 hydrogeological parameters have been considered for this study, and the final groundwater vulnerability map has been prepared by overlay weighted method with the help of DRASTIC index, which is classified into 5 vulnerable classes (very high, high, moderate, low, and very low). In the south and south-eastern regions of the basin namely Deghi, Bankada, Baram, Macha, Katin, Tilabani high groundwater contamination is been observed. For validating the model, the water quality parameters-nitrate and TDS have been used with the accuracy of 89% and 86% respectively. Using effective as well as scientifically approved methods, the anthropogenic and agricultural contamination can be controlled and managed which will lower the risk of contamination. This map can be further utilized as a base map for management of groundwater pollution and its planning.

Cleaning the river Damodar (India): impact of COVID-19 lockdown on water quality and future rejuvenation strategies.

Globally, it is established that the partial lockdown system assists to improve the health of the total environment due to inadequate anthropogenic actions in different economic sectors. The ample research on fitness of environment has been proved that the strict imposition of lockdown was the blessings of environment. The river Damodar has historical significance and lifeline for huge population of Jharkhand and West Bengal state of India but in the recent years the water quality has been deteriorated due to untreated industrial effluents and urban sewage. The main objective of this study is to examine the water quality of river Damodar during and prelockdown phase for domestic use and restoration of river ecosystem. A total of eleven (11) effluent discharge sites were selected in prelockdown and during lockdown phase. A new approach of water quality assessment, i.e., water pollution index (WPI) has been applied in this study. WPI is weightage free, unbiased method to analysis of water quality. The result shows that the physical, chemical and heavy elements were found beyond the standard limit in prelockdown period. The cation and anion were arranged in an order of Na2+ > K+ > Ca2+ > Mg2+ and Cl- > So4 - > No3 - > F- in both the sessions. WPI of prelockdown showed that about 100% water samples are of highly polluted. WPI of lockdown period showed that around 90.90% samples improved to 'good quality' and 9.10% of samples are of 'moderately polluted.' Hypothesis testing by 't' test proved that there was a significant difference (ρ = 0.05%) in values of each parameter between two periods. Null hypothesis was rejected and indicated the improvement of river water quality statistically. Spatial mapping using Arc GIS 10.4 interpolation (IDW) helps to understand spatial intensity of pollution load in two periods. This research study should be helpful for further management and spatial diagnosis of water resource of river Damodar.

Eco-restoration of river water quality during COVID-19 lockdown in the industrial belt of eastern India.

The sudden lockdown recovers the health of the total environment particularly air and water while the country's economic growth and socio-cultural tempo of people have been completely hampered due to the COVID-19 pandemic. Most of the industries within the catchment area of river Damodar have been closed; as a result, significant changes have been reflected throughout the stretch of river Damodar. The main objective of the study is to analyze the impact of lockdown on the water quality of river Damodar. A total of 55 samples was collected from eleven different confluence sites of nallas with the main river channel during and pre-lockdown period. The relevant methods like WQI, TSI, Pearson's correlation coefficient, and "t" test have been applied to evaluate the physical, chemical, and biological status of river water. The result of "t" test indicated that there are significant differences (α = 0.05) of each parameter between pre and during lockdown. Water quality index (WQI) is used for analysis of drinking water quality suitability followed by BIS. The values of WQI showed "very poor" (S1, S2, S3, S6, S7, and S11) to "unfit for drinking" (S4, S5, S8, S9, and S10) of river water during pre-monsoon season. The nutrient enrichment status of the river was analyzed by Trophic State Index (TSI) method and it shows the "High" eutrophic condition with a heavy concentration of algal blooms in almost an entire stretch. During lockdown, nutrient supplies like TN and TP have been reduced and is designated as "Low" (S1, S2) to "Moderate" (S3 to S11) eutrophic condition of middle stretch of Damodar. This research output of river Damodar will definitely assist to policy makers for sustainable environmental management despite the dilemma between development and conservation.

Regulation of Epithelial-to-Mesenchymal Transition by Alternative Translation Initiation Mechanisms and Its Implications for Cancer Metastasis.

Translation initiation plays a critical role in the regulation of gene expression for development and disease conditions. During the processes of development and disease, cells select specific mRNAs to be translated by controlling the use of diverse translation initiation mechanisms. Cells often switch translation initiation from a cap-dependent to a cap-independent mechanism during epithelial-to-mesenchymal transition (EMT), a process that plays an important role in both development and disease. EMT is involved in tumor metastasis because it leads to cancer cell migration and invasion, and is also associated with chemoresistance. In this review we will provide an overview of both the internal ribosome entry site (IRES)-dependent and N6-methyladenosine (m6A)-mediated translation initiation mechanisms and discuss how cap-independent translation enables cells from primary epithelial tumors to achieve a motile mesenchymal-like phenotype, which in turn drives tumor metastasis.

Cellular stress orchestrates the localization of hnRNP H to stress granules.

Heterogeneous nuclear ribonucleoprotein (hnRNP) H is a member of hnRNP H/F protein subfamily of hnRNPs that regulate the maturation and post-transcriptional processing of pre-mRNA. As a component of an mRNA export complex, hnRNP H shuttles mature mRNA from the nucleus to the cytoplasm. Although hnRNP H is primarily a nuclear protein, it can accumulate in the cytoplasm in certain tissues and cell types; however, the physiological relevance of hnRNP H cytoplasmic accumulation is unknown. Here we show that under cellular stress hnRNP H accumulates in the cytoplasm and is required for efficient recovery from cellular stress. Moreover, we find that cytoplasmic hnRNP H localizes to stress granules and that the RRM3 domain of hnRNP H is necessary for this localization. Together, our results demonstrate that hnRNP H accumulates in the cytoplasm under cellular stress and is recruited to stress granules.

microRNA-181a downregulates deptor for TGFß-induced glomerular mesangial cell hypertrophy and matrix protein expression.

TGFß contributes to mesangial cell hypertrophy and matrix protein increase in various kidney diseases including diabetic nephropathy. Deptor is an mTOR-interacting protein and suppresses mTORC1 and mTORC2 activities. We have recently shown that TGFß-induced inhibition of deptor increases the mTOR activity. The mechanism by which TGFß regulates deptor expression is not known. Here we identify deptor as a target of the microRNA-181a. We show that in mesangial cells, TGFß increases the expression of miR-181a to downregulate deptor. Decrease in deptor augments mTORC2 activity, resulting in phosphorylation/activation of Akt kinase. Akt promotes inactivating phosphorylation of PRAS40 and tuberin, leading to stimulation of mTORC1. miR-181a-mimic increased mTORC1 and C2 activities, while anti-miR-181a inhibited them. mTORC1 controls protein synthesis via phosphorylation of translation initiation and elongation suppressors 4EBP-1 and eEF2 kinase. TGFß-stimulated miR-181a increased the phosphorylation of 4EBP-1 and eEF2 kinase, resulting in their inactivation. miR-181a-dependent inactivation of eEF2 kinase caused dephosphorylation of eEF2. Consequently, miR-181a-mimic increased protein synthesis and hypertrophy of mesangial cells similar to TGFß. Anti-miR-181a blocked these events in a deptor-dependent manner. Finally, TGFß-miR-181a-driven deptor downregulation increased the expression of fibronectin. Our results identify a novel mechanism involving miR-181a-driven deptor downregulation, which contributes to mesangial cell pathologies in renal complications.

Reciprocal regulation of miR-214 and PTEN by high glucose regulates renal glomerular mesangial and proximal tubular epithelial cell hypertrophy and matrix expansion.

Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3ß, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells.

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression.

High glucose milieu inhibits PTEN expression to activate Akt kinase and induces glomerular mesangial cell hypertrophy and matrix protein expression in diabetic nephropathy. Specific mechanism by which high glucose inhibits PTEN expression is not clear. We found that high glucose increased the expression of the microRNA-26a (miR-26a) in mesangial cells. Using a sensor plasmid with 3'UTR-driven luciferase, we showed PTEN as a target of miR-26a in response to high glucose. Overexpression of miR-26a reduced the PTEN protein levels resulting in increased Akt kinase activity similar to high glucose treatment. In contrast, anti-miR-26a reversed high glucose-induced suppression of PTEN with concomitant inhibition of Akt kinase activity. Akt-mediated phosphorylation of tuberin and PRAS40 regulates mTORC1, which is necessary for mesangial cell hypertrophy and matrix protein expression. Inhibition of high glucose-induced miR-26a blocked phosphorylation of tuberin and PRAS40, which lead to suppression of phosphorylation of S6 kinase and 4EBP-1, two substrates of mTORC1. Furthermore, we show that expression of miR-26a induced mesangial cell hypertrophy and increased fibronectin and collagen I (α2) expression similar to that observed with the cells incubated with high glucose. Anti-miR-26a inhibited these phenomena in response to high glucose. Together our results provide the first evidence for the involvement of miR-26a in high glucose-induced mesangial cell hypertrophy and matrix protein expression. These data indicate the potential therapeutic utility of anti-miR-26a for the complications of diabetic kidney disease.

Changing bacterial profile of Sundarbans, the world heritage mangrove: impact of anthropogenic interventions.

Mangrove microbial communities and their associated activities have profound impact on biogeochemical cycles. Although microbial composition and structure are known to be influenced by biotic and abiotic factors in the mangrove sediments, finding direct correlations between them remains a challenge. In this study we have explored sediment bacterial diversity of the Sundarbans, a world heritage site using a culture-independent molecular approach. Bacterial diversity was analyzed from three different locations with a history of exposure to differential anthropogenic activities. 16S rRNA gene libraries were constructed and partial sequencing of the clones was performed to identify the microbial strains. We identified bacterial strains known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbon degradation, and heavy metal resistance. Canonical Correspondence Analysis of the environmental and exploratory datasets revealed correlations between the ecological indices associated with pollutant levels and bacterial diversity across the sites. Our results indicate that sites with similar exposure of anthropogenic intervention reflect similar patterns of microbial diversity besides spatial commonalities.

Improvement of degummed ramie fiber properties upon treatment with cellulase secreting immobilized A. larrymoorei A1.

Degummed ramie fiber is widely used in the textile industry. Cellulase enzyme can be effectively used for bio-polishing of the ramie fiber. We immobilized Agrobacterium larrymoorei A1, a potent extra-cellular cellulase producing bacteria, in Ca-alginate. The production of enzyme significantly increased with increasing alginate concentration and reached a maximum activity of 0.28 IU/ml at 20 g/l, which was 32% higher as compared to free cells. These immobilized cells were used on ramie fibers. Scanning electron micrograph (SEM) and differential interference contrast (DIC) studies showed increased smoothness and orientation of surface structure of the fibers after 19.5 h. The single fiber tenacity was almost same as compared to non-treated fiber and the initial modulus increased by 24.01%. The remarkable reusability of these immobilized cells provides a cost effective method for treatment of natural fibers containing cellulose.

High glucose forces a positive feedback loop connecting Akt kinase and FoxO1 transcription factor to activate mTORC1 kinase for mesangial cell hypertrophy and matrix protein expression.

High glucose-induced Akt acts as a signaling hub for mesangial cell hypertrophy and matrix expansion, which are recognized as cardinal signatures for the development of diabetic nephropathy. How mesangial cells sustain the activated state of Akt is not clearly understood. Here we show Akt-dependent phosphorylation of the transcription factor FoxO1 by high glucose. Phosphorylation-deficient, constitutively active FoxO1 inhibited the high glucose-induced phosphorylation of Akt to suppress the phosphorylation/inactivation of PRAS40 and mTORC1 activity. In contrast, dominant negative FoxO1 increased the phosphorylation of Akt, resulting in increased mTORC1 activity similar to high glucose treatment. Notably, FoxO1 regulates high glucose-induced protein synthesis, hypertrophy, and expression of fibronectin and PAI-1. High glucose paves the way for complications of diabetic nephropathy through the production of reactive oxygen species (ROS). We considered whether the FoxO1 target antioxidant enzyme catalase contributes to sustained activation of Akt. High glucose-inactivated FoxO1 decreases the expression of catalase to increase the production of ROS. Moreover, we show that catalase blocks high glucose-stimulated Akt phosphorylation to attenuate the inactivation of FoxO1 and PRAS40, resulting in the inhibition of mTORC1 and mesangial cell hypertrophy and fibronectin and PAI-1 expression. Finally, using kidney cortices from type 1 diabetic OVE26 mice, we show that increased FoxO1 phosphorylation is associated with decreased catalase expression and increased fibronectin and PAI-1 expression. Together, our results provide the first evidence for the presence of a positive feedback loop for the sustained activation of Akt involving inactivated FoxO1 and a decrease in catalase expression, leading to increased ROS and mesangial cell hypertrophy and matrix protein expression.

TGFß-induced deptor suppression recruits mTORC1 and not mTORC2 to enhance collagen I (α2) gene expression.

Enhanced TGFß activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFß rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFß treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFß reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFß treatment. Importantly, downregulation of deptor by TGFß increased the expression of Hif1α by increasing translation of its mRNA. TGFß-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFß-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFß-induced renal fibrosis.

microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKß-mTORC1 axis to regulate renal cancer cell invasion.

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKß, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKß blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKß. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4 and dominant negative IKKß. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor-PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells.

A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF.

Platelet-derived growth factor BB and its receptor (PDGFRß) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.

NFκB-mediated cyclin D1 expression by microRNA-21 influences renal cancer cell proliferation.

MicroRNAs regulate post-transcriptomic landscape in many tumors including renal cell carcinoma. We have recently shown significantly increased expression of miR-21 in renal tumors and that this miRNA contributes to the proliferation of renal cancer cells in culture. However, the mechanism by which miR-21 regulates renal cancer cell proliferation is poorly understood. Addiction to constitutive NFκB activity is hallmark of many cancers including renal cancer. Using miR-21 Sponge in renal cancer cells to block endogenous function of miR-21, we show inhibition of phosphorylation of p65 subunit of NFκB, IKKß and IκB, which results in attenuation of NFκB transcriptional activity. Subtle reduction in the tumor suppressor PTEN has been linked to various malignancies. We showed previously that miR-21 targeted PTEN in renal cancer cells. Inhibition of PTEN by siRNAs restored miR-21 Sponge-induced suppression of phosphorylation of p65, IKKß, IκB and NFκB transcriptional activity along with reversal of miR-21 Sponge-reduced phosphorylation of Akt. Expression of constitutively active Akt protected against miR-21 Sponge- and PTEN-mediated decrease in p65/IKKß/IκB phosphorylation and NFκB transcriptional activity. Furthermore, IKKß and p65 were required for miR-21-induced renal cancer cell proliferation. Interestingly, miR-21 controlled the expression of cyclin D1 through NFκB-dependent transcription. Finally, we demonstrate that miR-21-regulated renal cancer cell proliferation is mediated by cyclin D1 and CDK4. Together, our results establish a molecular order of a phosphatase-kinase couple involving PTEN/Akt/IKKß and NFκB-dependent cyclin D1 expression for renal carcinoma cell proliferation by increased miR-21 levels.