The paracrine isthmin1 transcriptionally regulated by C/EBPß exacerbates pulmonary vascular leakage in murine sepsis.

It is known that pulmonary vascular leakage, a key pathological feature of sepsis-induced lung injury, is largely regulated by perivascular cells. However, the underlying mechanisms have not been fully uncovered. In the present study, we aimed to evaluate the role of isthmin1, a secretory protein originating from alveolar epithelium, in the pulmonary vascular leakage during sepsis and to investigate the regulatory mechanisms of isthmin1 gene transcription. We observed an elevated isthmin1 gene expression in the pulmonary tissue of septic mice induced by cecal ligation and puncture (CLP), as well as in primary murine alveolar type II epithelial cells (ATII) exposed to lipopolysaccharide (LPS). Furthermore, we confirmed that isthmin1 derived from ATII contributes to pulmonary vascular leakage during sepsis. Specifically, adenovirus-mediated isthmin1 disruption in ATII led to a significant attenuation of the increased pulmonary microvascular endothelial cell (PMVEC) hyperpermeability in a PMVEC/ATII coculture system when exposed to LPS. In addition, adeno-associated virus 9 (AAV9)-mediated knockdown of isthmin1 in the alveolar epithelium of septic mice significantly attenuated pulmonary vascular leakage. Finally, mechanistic studies unveiled that nuclear transcription factor CCAAT/enhancer binding protein (C/EBP)ß participates in isthmin1 gene activation by binding directly to the cis-regulatory element of isthmin1 locus and may contribute to isthmin1 upregulation during sepsis. Collectively, the present study highlighted the impact of the paracrine protein isthmin1, derived from ATII, on the exacerbation of pulmonary vascular permeability in sepsis and revealed a new regulatory mechanism for isthmin1 gene transcription.NEW & NOTEWORTHY This article addresses the role of the alveolar epithelial-secreted protein isthmin1 on the exacerbation of pulmonary vascular permeability in sepsis and identified nuclear factor CCAAT/enhancer binding protein (C/EBP)ß as a new regulator of isthmin1 gene transcription. Targeting the C/EBPß-isthmin1 regulatory axis on the alveolar side would be of great value in the treatment of pulmonary vascular leakage and lung injury induced by sepsis.

Activation of brain lactate receptor GPR81 aggravates exercise-induced central fatigue.

Exercise-induced fatigue is a complex physiological phenomenon and is greatly influenced by central mechanisms in brain. As one of the most abundant circulating carbon metabolites, l-lactate in brain has been considered to be an important supplementary fuel during exercise; however, whether it plays a signaling role in fatigue remains largely obscure. In this study, our results initially revealed that brain l-lactate levels were increased after an exhaustive swimming session in several brain regions including motor cortex, hippocampus, and cerebellum. Then, we examined the specific role of brain lactate receptor, also known as hydroxycarboxylic acid receptor 1 (GPR81), in exercise-induced fatigue. We found that intracerebroventricular injection of either d-lactate (an enantiomer that could mediate activation of GPR81 as l-lactate) or a potent GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA), significantly decreased the swimming time to fatigue. After being subjected to the same weight-loaded swimming for 30 min, no obvious changes of blood lactate levels, gastrocnemius pAMPK/AMPK ratio, and glycogen contents were observed between intracerebroventricular CHBA-injected mice and vehicle-treated ones, which suggested a comparable degree of peripheral fatigue. Meanwhile, there were higher extracellular γ-aminobutyric acid (GABA) levels and lower extracellular glutamate levels and glutamate/GABA ratio in motor cortex of the intracerebroventricular CHBA-injected mice than that of vehicle-treated ones, indicating a greater extent of central fatigue in CHBA-injected mice than that in vehicle animals. Collectively, our results suggested that an increased level of brain l-lactate acts as a signaling molecule via activating GPR81, which in turn exacerbates central fatigue during exercise.

Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

HIF1α-induced upregulation of KLF4 promotes migration of human vascular smooth muscle cells under hypoxia.

Hypoxia-induced vascular smooth muscle cells (VSMCs) migration plays an important role in vascular remodeling and is implicated in vascular diseases, such as atherosclerosis and pulmonary hypertension. We previously observed the increased expression of krüppel-like factor 4 (KLF4) in VSMCs under hypoxia. However, whether the upregulation of KLF4 participates in hypoxia-induced VSMCs migration is still unknown. In this study, we demonstrated that KLF4 was an important player in the process of VSMCs migration under hypoxia since interference of KLF4 by small interfering RNA mostly dampened hypoxia-induced migration of VSMCs. In addition, using luciferase reporter and ChIP assays, we confirmed two hypoxia-inducible factor 1α (HIF1α) binding elements (located at -150 to -163 and -3922 to -3932) in the upstream regulatory region of klf4 locus and identified KLF4 as a novel direct target gene of HIF1α. Our findings unveil a novel regulatory mechanism that involves HIF1α-induced upregulation of KLF4, which plays a vital role in VSMCs migration under hypoxia.

Assessment of fatigue-related biochemical alterations in a rat swimming model under hypoxia.

It is well known that exercise-induced fatigue is exacerbated following hypoxia exposure and may arise from central and/or peripheral mechanisms. To assess the relative contribution of peripheral and central factors to exercise-induced fatigue under hypoxia, a rat model of fatigue by a bout of exhaustive swimming was established and fatigue-related biochemical changes in normoxic and severe hypoxic conditions were compared. Rats were randomly divided into four groups: normoxia resting (NR), exhaustive swimming (NE), hypoxia resting (HR) and exhaustive swimming (HE). The swimming time to exhaustion with a weight equal to 2.5% of their body weight reduced under hypoxia. There were lower blood lactate levels, lower gastrocnemius pAMPK/AMPK ratios and higher gastrocnemius glycogen contents in the HE than in the NE groups, which all suggested a lower degree of peripheral fatigue in the HE group than in the NE group. Meanwhile, there was a significant increase in striatal 3,4-dihydroxyphenylacetic acid (DOPAC) caused by exhaustive swimming under normoxia, whereas this increase was almost blunted under severe hypoxia, indicating that hypoxia might exacerbate exercise-induced central fatigue. These biochemical changes suggest that from normoxia to severe hypoxia, the relative contribution of peripheral and central factors to exercise-induced fatigue alters, and central fatigue may play a predominant role in the decline in exercise performance under hypoxia.

EGF-induced C/EBPß participates in EMT by decreasing the expression of miR-203 in esophageal squamous cell carcinoma cells.

Epithelial-mesenchymal transition (EMT) is a developmental program that is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBPß has been reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here, we report for the first time, that the truncated CCAAT-enhancer-binding protein ß (C/EBPß) LIP isoform is abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBPß LIP mediates epithelial growth factor (EGF)-induced EMT and increases migration and invasion of esophageal cancer cells in a manner that is dependent on miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBPß LIP. Disruption of C/EBPß LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBPß LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBPß LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a new regulatory mechanism that involves C/EBPß-LIP-mediated downregulation of miR-203, which plays a key role in EMT and metastasis.

HIF-1 alpha-induced up-regulation of miR-9 contributes to phenotypic modulation in pulmonary artery smooth muscle cells during hypoxia.

Pulmonary artery smooth muscle cells (PASMCs) are associated with the development of hypoxic pulmonary hypertension (HPH). Recent studies have implicated a critical role for microRNAs (miRNAs) in HPH; however, their expression and regulation in hypoxia-mediated phenotypic modulation of PASMCs remains largely unclear. Here, we report that miR-9 was induced in hypoxia and involved in a hypoxia-induced phenotypic switch in rat primary PASMCs. Knockdown of miR-9 followed by hypoxia exposure attenuated PASMCs proliferation and enhanced the expression of contractile genes in vascular smooth muscle cells (VSMCs), while overexpression of miR-9 in normoxia promoted a proliferative phenotype in PASMCs. The primary transcripts of miR-9-1 and miR-9-3, but not miR-9-2, increased dramatically after hypoxia, whereas silencing of the hypoxia-associated transcription factor HIF-1α following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF-1α binding motifs on miR-9-1 and one motif on miR-9-3 located within the 5-kb region upstream of the transcriptional start sites. Chromatin immunoprecipitation assay revealed that hypoxia enhanced the direct interaction between HIF-1α and the regulatory elements of miR-9-1 and miR-9-3. Reporter assays showed that the regulatory regions of miR-9-1 and miR-9-3 behaved as enhancers in a HIF-1α-dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF-1α-mediated up-regulation of miR-9, which plays a role in the hypoxia-induced phenotypic switch of PASMCs.

Transcriptional regulation of miR-146b by C/EBPß LAP2 in esophageal cancer cells.

Recent clinical study indicated that up-regulation of miR-146b was associated with poor overall survival of patients in esophageal squamous cell carcinoma. However, the underlying mechanism of miR-146b dysregulation remains to be explored. Here we report that miR-146b promotes cell proliferation and inhibits cell apoptosis in esophageal cancer cell lines. Mechanismly, two C/EBPß binding motifs are located in the miR-146b promoter conserved region. Among the three isoforms of C/EBPß, C/EBPß LAP2 positively regulated miR-146b expression and increases miR-146b levels in a dose-dependent manner through transcription activation of miR-146b gene. Together, these results suggest a miR-146b regulatory mechanism involving C/EBPß, which may contribute to the up-regulation of miR-146b in esophageal squamous cell carcinoma.

Cerebral Lactate Participates in Hypoxia-induced Anapyrexia Through its Receptor G Protein-coupled Receptor 81.

Hypoxia-induced anapyrexia is thought to be a regulated decrease in body core temperature (Tcore), but the underlying mechanism remains unclear. Recent evidence suggests that lactate, a glycolysis product, could modulate neuronal excitability through the G protein-coupled receptor 81 (GPR81). The present study aims to elucidate the role of central lactate and GPR81 in a rat model of hypoxia-induced anapyrexia. The findings revealed that hypoxia (11.1% O2, 2 h) led to an increase in lactate in cerebrospinal fluid (CSF) and a decrease in Tcore. Injection of dichloroacetate (DCA, 5 mg/kg, 1 µL), a lactate production inhibitor, to the third ventricle (3 V), alleviated the increase in CSF lactate and the decrease in Tcore under hypoxia. Immunofluorescence staining showed GPR81 was expressed in the preoptic area of hypothalamus (PO/AH), the physiological thermoregulation integration center. Under normoxia, injection of GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA, 0.05 mg/kg, 1 µL) to the 3 V, reduced Tcore significantly. In addition, hypoxia led to a dramatic increase in tail skin temperature and a decrease in interscapular brown adipose tissue skin temperature. The number of c-Fos+ cells in the PO/AH increased after exposure to 11.1% O2 for 2 h, but administration of DCA to the 3 V blunted this response. Injection of CHBA to the 3 V also increased the number of c-Fos+ cells in the PO/AH under normoxia. In light of these, our research has uncovered the pivotal role of central lactate-GPR81 signaling in anapyrexia, thereby providing novel insights into the mechanism of hypoxia-induced anapyrexia.

Identification of a Hippocampus-to-Zona Incerta Projection involved in Motor Learning.

Motor learning (ML), which plays a fundamental role in growth and physical rehabilitation, involves different stages of learning and memory processes through different brain regions. However, the neural mechanisms that underlie ML are not sufficiently understood. Here, a previously unreported neuronal projection from the dorsal hippocampus (dHPC) to the zona incerta (ZI) involved in the regulation of ML behaviors is identified. Using recombinant adeno-associated virus, the projections to the ZI are surprisingly identified as originating from the dorsal dentate gyrus (DG) and CA1 subregions of the dHPC. Furthermore, projection-specific chemogenetic and optogenetic manipulation reveals that the projections from the dorsal CA1 to the ZI play key roles in the acquisition and consolidation of ML behaviors, whereas the projections from the dorsal DG to the ZI mediate the retrieval/retention of ML behaviors. The results reveal new projections from the dorsal DG and dorsal CA1 to the ZI involved in the regulation of ML and provide insight into the stages over which this regulation occurs.

Hypoxia-Induced Myocardial Hypertrophy Companies with Apoptosis Enhancement and p38-MAPK Pathway Activation.

Li, Xiaoxu, Zhijun Pu, Gang Xu, Yidong Yang, Yu Cui, Xiaoying Zhou, Chenyuan Wang, Zhifeng Zhong, Simin Zhou, Jun Yin, Fabo Shan, Chengzhong Yang, Li Jiao, Dewei Chen, and Jian Huang. Hypoxia-induced myocardial hypertrophy companies with apoptosis enhancement and p38-MAPK pathway activation. High Alt Med Biol. 00:00-00, 2024. Background: Right ventricular function and remodeling are closely associated with symptom severity and patient survival in hypoxic pulmonary hypertension. However, the detailed molecular mechanisms underlying hypoxia-induced myocardial hypertrophy remain unclear. Methods: In Sprague-Dawley rats, hemodynamics were assessed under both normoxia and hypobaric hypoxia at intervals of 7 (H7), 14 (H14), and 28 (H28) days. Morphological changes in myocardial tissue were examined using hematoxylin and eosin (HE) staining, while myocardial hypertrophy was evaluated with wheat germ agglutinin (WGA) staining. Apoptosis was determined through TUNEL assays. To further understand the mechanism of myocardial hypertrophy, RNA sequencing was conducted, with findings validated via Western blot analysis. Results: The study demonstrated increased hypoxic pulmonary hypertension and improved right ventricular diastolic and systolic function in the rat models. Significant elevations in pulmonary arterial systolic pressure (PASP), mean pulmonary arterial pressure (mPAP), right ventricular mean pressure (RVMP), and the absolute value of +dp/dtmax were observed in the H14 and H28 groups compared with controls. In addition, right ventricular systolic pressure (RVSP), -dp/dtmax, and the mean dp/dt during isovolumetric relaxation period were notably higher in the H28 group. Heart rate increased in the H14 group, whereas the time constant of right ventricular isovolumic relaxation (tau) was reduced in both H14 and H28 groups. Both the right heart hypertrophy index and the heart weight/body weight ratio (HW/BW) were elevated in the H14 and H28 groups. Myocardial cell cross-sectional area also increased, as shown by HE and WGA staining. Western blot results revealed upregulated HIF-1α levels and enhanced HIF-2α expression in the H7 group. In addition, phosphorylation of p38 and c-fos was augmented in the H28 group. The H28 group showed elevated levels of Cytochrome C (Cyto C), whereas the H14 and H28 groups exhibited increased levels of Cleaved Caspase-3 and the Bax/Bcl-2 ratio. TUNEL analysis revealed a rise in apoptosis with the extension of hypoxia duration in the right ventricle. Conclusions: The study established a link between apoptosis and p38-MAPK pathway activation in hypoxia-induced myocardial hypertrophy, suggesting their significant roles in this pathological process.

[Effects of acute hypobaric hypoxia and exhaustive exercise on AMP-activated protein kinase phosphorylation in rat skeletal muscle].

The present study was aimed to explore the changes of phosphorylated AMP-activated protein kinase (pAMPK) level in skeletal muscle after exposure to acute hypobaric hypoxia and exhaustive exercise. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sea level and high altitude groups. The rats in high altitude group were submitted to simulated 5 000 m of high altitude in a hypobaric chamber for 24 h, and sea level group was maintained at normal conditions. All the rats were subjected to exhaustive swimming exercise. The exhaustion time was recorded. Before and after the exercise, blood lactate and glycogen content in skeletal muscle were determined; AMPK and pAMPK levels in skeletal muscle were detected by Western blot. The results showed that the exhaustion time was significantly decreased after exposure to high altitude. At the moment of exhaustion, high altitude group had lower blood lactate concentration and higher surplus glycogen content in gastrocnemius compared with sea level group. Exhaustive exercise significantly increased the pAMPK/AMPK ratio in rat skeletal muscles from both sea level and high altitude groups. However, high altitude group showed lower pAMPK/AMPK ratio after exhaustion compared to sea level group. These results suggest that, after exposure to acute hypobaric hypoxia, the decrement in exercise capacity may not be due to running out of glycogen, accumulation of lactate or disturbance in energy status in skeletal muscle.

Inhibition of HIF-1α-AQP4 axis ameliorates brain edema and neurological functional deficits in a rat controlled cortical injury (CCI) model.

Traumatic brain injury (TBI) is an important cause of death in young adults and children. Till now, the treatment of TBI in the short- and long-term complications is still a challenge. Our previous evidence implied aquaporin 4 (AQP4) and hypoxia inducible factor-1α (HIF-1α) might be potential targets for TBI. In this study, we explored the roles of AQP4 and HIF-1α on brain edema formation, neuronal damage and neurological functional deficits after TBI using the controlled cortical injury (CCI) model. The adult male Sprague Dawley rats were randomly divided into sham and TBI group, the latter group was further divided into neutralized-AQP4 antibody group, 2-methoxyestradiol (2-ME2) group, and their corresponding control, IgG and isotonic saline groups, respectively. Brain edema was examined by water content. Hippocampal neuronal injury was assessed by neuron loss and neuronal skeleton related protein expressions. Spatial learning and memory deficits were evaluated by Morris water maze test and memory-related proteins were detected by western blot. Our data showed that increased AQP4 protein level was closely correlated with severity of brain edema after TBI. Compared with that in the control group, both blockage of AQP4 with neutralized-AQP4 antibody and inhibition of HIF-1α with 2-ME2 for one-time treatment within 30-60 min post TBI significantly ameliorated brain edema on the 1st day post-TBI, and markedly alleviated hippocampal neuron loss and spatial learning and memory deficits on the 21st day post-TBI. In summary, our preliminary study revealed the short-term and long-term benefits of targeting HIF-1α-AQP4 axis after TBI, which may provide new clues for the selection of potential therapeutic targets for TBI in clinical practice.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.

NLRP3 Inflammasome-Dependent Increases in High Mobility Group Box 1 Involved in the Cognitive Dysfunction Caused by Tau-Overexpression.

Tau hyperphosphorylation is a characteristic alteration present in a range of neurological conditions, such as traumatic brain injury (TBI) and neurodegenerative diseases. Treatments targeting high-mobility group box protein 1 (HMGB1) induce neuroprotective effects in these neuropathologic conditions. However, little is known about the interactions between hyperphosphorylated tau and HMGB1 in neuroinflammation. We established a model of TBI with controlled cortical impacts (CCIs) and a tau hyperphosphorylation model by injecting the virus encoding human P301S tau in mice, and immunofluorescence, western blotting analysis, and behavioral tests were performed to clarify the interaction between phosphorylated tau (p-tau) and HMGB1 levels. We demonstrated that p-tau and HMGB1 were elevated in the spatial memory-related brain regions in mice with TBI and tau-overexpression. Animals with tau-overexpression also had significantly increased nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation, which manifested as increases in apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), activating caspase-1 and interleukin 1 beta (IL-1ß) levels. In addition, NLRP3-/- mice and the HMGB1 inhibitor, glycyrrhizin, were used to explore therapeutic strategies for diseases with p-tau overexpression. Compared with wild-type (WT) mice with tau-overexpression, downregulation of p-tau and HMGB1 was observed in NLRP3-/- mice, indicating that HMGB1 alterations were NLRP3-dependent. Moreover, treatment with glycyrrhizin at a late stage markedly reduced p-tau levels and improved performance in the Y- and T-mazes and the ability of tau-overexpressing mice to build nests, which revealed improvements in spatial memory and advanced hippocampal function. The findings identified that p-tau has a triggering role in the modulation of neuroinflammation and spatial memory in an NLRP3-dependent manner, and suggest that treatment with HMGB1 inhibitors may be a better therapeutic strategy for tauopathies.

Effects of long term low- and high-dose sodium arsenite exposure in human transitional cells.

Epidemiological studies have revealed the association between increased risk of bladder cancer and chronic arsenic exposure. Here, we explored biological effects of arsenic in T24. Microarray analysis was applied to analyze mRNA in T24 following 0, 2 or 5 µM sodium arsenite (As) exposure for 72 hours. Long term (up to 140 days) low-dose (200 nM) and high-dose (1,000 nM) As decreased E-cadherin protein level through different mechanisms because the mRNA levels of E-cadherin increased following low-dose As exposure but decreased following high-dose As exposure. Long term As increased the protein levels of N-cadherin, vimentin, ß-catenin, and slug. Low-dose As exposure resulted in a change in the morphology of T24 cells from an epithelial to a mesenchymal-like appearance. Knockdown of E-cadherin increased the protein levels of N-cadherin, vimentin, ß-catenin, and slug. Cell proliferation and growth of T24 with or without As exposure for 100 days were assayed using EdU and WST, respectively. Low-dose As exposure increased cell proliferation and growth while high-dose As exposure decreased both. Long term As activated p53 on account of increasing protein levels of p53, p-p53 (Ser15), and mRNA levels of p21. These demonstrate that arsenic exposure exerts multiple effects. Long term low- or high-dose arsenic induces epithelial-mesenchymal transition, likely via downregulation of E-cadherin, activates p53, and differently affects cell proliferation/growth.