Autophagic Vacuolation Induced by Excess ROS Generation in HABP1/p32/gC1qR Overexpressing Fibroblasts and Its Reversal by Polymeric Hyaluronan

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca⁺⁺ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07.     

HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal alpha helix

Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domain of HABP1, its N- and C-terminal truncated variants (DeltaN.HABP1 and DeltaC.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, DeltaC.HABP1 too directly interacts with CDC 25 while DeltaN.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal alpha-helix.     

Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to vascular endothelium and for platelet-mediated clumping

The ability of Plasmodium falciparum-infected red blood cells (IRBCs) to bind to vascular endothelium, thus enabling sequestration in vital host organs, is an important pathogenic mechanism in malaria. Adhesion of P. falciparum IRBCs to platelets, which results in the formation of IRBC clumps, is another cytoadherence phenomenon that is associated with severe disease. Here, we have used in vitro cytoadherence assays to demonstrate, to our knowledge for the first time, that P. falciparum IRBCs use the 32-kDa human protein gC1qR/HABP1/p32 as a receptor to bind to human brain microvascular endothelial cells. In addition, we show that P. falciparum IRBCs can also bind to gC1qR/HABP1/p32 on platelets to form clumps. Our study has thus identified a novel host receptor that is used for both adhesion to vascular endothelium and platelet-mediated clumping. Given the association of adhesion to vascular endothelium and platelet-mediated clumping with severe disease, adhesion to gC1qR/HABP1/p32 by P. falciparum IRBCs may play an important role in malaria pathogenesis.     

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased HA pool, formation of the HA cable structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.     

Constitutive expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR) in normal fibroblast cells perturbs its growth characteristics and induces apoptosis

Hyaluronan binding protein 1 (HABP1) is a ubiquitously expressed multifunctional phospho-protein that interacts with a wide range of ligands and is implicated in cell signalling. Recently, we have reported that HABP1 is an endogenous substrate for MAP kinase and upon mitogenic stimulation it is translocated to the nucleus in a MAP kinase-dependent manner (Biochem. Biophys. Res. Commun. 291(4) (2002) 829-837). This prompted us to investigate the role of HABP1 in cell growth or otherwise in low MAP kinase background. We demonstrate that HABP1, when overexpressed in normal rat skin fibroblasts, remained in the cytosol, primarily concentrated around the nuclear periphery. However, HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, which was corrected by frequent medium replenishment. Further investigation revealed that HABP1 overexpressing cells undergo apoptosis, as detected by TUNEL assay, induction of Bax expression, and FACS analysis, and they failed to enter into the S-phase. Periodic medium supplementation prevented these cells from undergoing apoptotic death. We also demonstrate that upon induction of apoptosis in HeLa cells by cisplatin, HABP1 level is upregulated, indicating a correlation between HABP1 and cell death in a normal cellular environment.     

Hyaluronan binding protein 1 (HABP1)/C1QBP/p32 is an endogenous substrate for MAP kinase and is translocated to the nucleus upon mitogenic stimulation

The role of hyaluronan binding protein 1 (HABP1) in cell signaling was investigated and in vitro kinase assay demonstrated that it is a substrate for MAP kinase. Phosphorylation of endogenous HABP1 was also observed following treatment of J774 cells with PMA. HABP1 was coimmunoprecipitated with activated ERK, confirming their physical interaction in the cellular context. Upon PMA stimulation of normal rat fibroblast (F111) and transformed (HeLa) cells, the HABP1 level in the cytoplasm gradually decreased with a parallel increase in the nucleus. In HeLa cells, within 6 h of PMA treatment, HABP1 was completely translocated to the nucleus, which was prevented by PD98059, a selective inhibitor of ERK. We also observed that the nuclear translocation of HABP1 is concurrent with that of ERK, suggesting that ERK activation is a requirement for the translocation of HABP1. It is thus established for the first time that HABP1 is a substrate for ERK and an integral part of the MAP kinase cascade.     

HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

Background

Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.

Methods

MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.

Results

HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT–PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.

Conclusion

These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.     

Daintain/AIF-1强化了HepG2细胞顺铂耐药性的产生

目的：探讨炎症因子Daintain/AIF-1对肝癌细胞耐药性产生的影响。方法：利用MTT法测定耐药HepG2细胞株的IC50,流式细胞术测定耐药细胞株期凋亡率,HPLC方法检测隔耐药细胞株胞内顺铂的外排。结果：Daintain/AIF-1提高了HepG2耐药细胞株的IC50;再次受到相同剂量顺铂的攻击时,Daintain/AIF-1与顺铂联合运用构建的耐药细胞株凋亡率明显下降;Daintain/AIF-1促进了耐药细胞株胞内顺铂的外排。结论：此研究表明Daintain/AIF-1通过影响胞内顺铂的外排而促进了肝癌细胞对顺铂耐药性的产生。     

HCV 1a/1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a/1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Western blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV)1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达.结果证明,转染细胞中检测到分子量约70 kDa的HCV NS3蛋白, 转染细胞连续传20代, 仍能检测到HCV正、负链RNA.表明该HCV嵌合体可以在细胞中复制和表达,HCV 1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制. HCV 5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合.3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失"A",9409,9410位及9495,9496,9497位分别插入"TT"和"AAT"可不影响RdRp的生物活性.本研究对阐明HCV复制和翻译机制有重要意义.     

HCV 1a／1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a／1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Westem blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV) 1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCV NS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RdRp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。     

脱氢表雄酮调节HepG2/GFP-HBx细胞p16基因甲基化及其与细胞周期和凋亡的相关性

目的探讨HBx与p16基因甲基化的关系,研究脱氢表雄酮（DHEA）对p16基因甲基化以及细胞周期和细胞凋亡的调节作用。方法以HepG2、HepG2/GFP和HepG2/GFP-HBx三种细胞为材料,采用MTT法检测细胞生长;流式细胞术检测细胞周期和凋亡率;MSP-PCR检测p16基因甲基化水平。比较分析HBx与p16基因启动子甲基化、DHEA与各检测指标的关系。结果HepG2/GFP-HBx细胞与HepG2细胞和HepG2/GFP细胞相比,细胞的增殖速度提高（P〈0.05）,G0/G1期细胞减少,S期细胞增多（P〈0.05）,凋亡率降低（P〈0.05）;HepG2/GFP-HBx细胞的p16基因启动子呈现高水平甲基化,HepG2和HepG2/GFP细胞呈现高水平非甲基化。100μmol/L的DHEA使三种细胞的增殖速度降低（P〈0.05）,G0/G1期细胞增加,S期细胞减少（P〈0.05）,凋亡率提高（P〈0.05）。DHEA可下调HepG2/GFP-HBx细胞的p16基因启动子甲基化水平,但对HepG2和HepG2/GFP细胞的p16基因启动子甲基化水平不产生影响。结论HBx引起肝癌细胞p16基因甲基化,并缩短细胞周期抑制细胞凋亡;DHEA可明显下调HBx引起的p16基因甲基化水平,延长细胞周期促进细胞凋亡,在无HBx基因整合的情况下,DHEA对肝癌细胞生长、细胞周期和凋亡的影响不通过p16基因甲基化的途径实现。     

Increased Diacylglycerol Levels Inhibit [20-3H]Phorbol 12, 13-Dibutyrate Binding and the Glucocorticoid-Mediated Increase in Glycerol Phosphate Dehydrogenase Levels in C6 Rat Glioma Cells

I examined whether the phorbol ester-mediated inhibition of glycerol 3-phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2-bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20-3H]phorbol 12,13-dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2-Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol 1-oleoyl 2-acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid-mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.     

Altered Tumor Biology and Tumorigenesis in Irradiated and Chemical Carcinogen-Treated Single and Combined Connexin32/p27Kip1-Deficient Mice

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical and radiation-induced liver and lung tumorigenesis. This increased tumor incidence is associated with altered tumor biology including enhanced tumor progression and an increased percent of MAPK-active tumors. Likewise, mice lacking the tumor suppressor/cell cycle regulator p27^Kip1 exhibit increased tumorigenesis in a variety of tissues following chemical and radiation induction. Interestingly, in a double-deficient mouse model (DKO), additional loss of p27^Kip1 in a Cx32-KO background results in attenuation of liver and lung tumorigenesis as well as MAPK activation profiles, suggesting pathway interaction. While these mouse strains exhibit altered liver and lung tumor susceptibility following both chemical (DEN) and radiation (X-ray) induction protocols, comparisons of the resulting tumor incidence, multiplicity, tumor progression, and MAPK activation in response to these two distinct carcinogens underscores the separate influence of each individual gene on both tumor formation and activation of specific oncogenic pathways. Furthermore, these studies demonstrate that different carcinogens interact disparately with Cx32/p27^Kip1 genotypic backgrounds in situ resulting in varied tumorigenic response.     

Down-Regulation of ID2-AS1 Alleviates the Neuronal Injury Induced by 1-Methy1-4-Phenylpyridinium in Human Neuroblastoma Cell Line SH-SY5Y Cells Through Regulating miR-199a-5p/IFNAR1/JAK2/STAT1 Axis

We aimed to illustrate the roles and molecular mechanisms of ID2-AS1 in parkinson’s disease (PD). Methods: qRT-PCR detected the expression of ID2-AS1. CCK-8, LDH release assays the effect of ID2-AS1 knockdown on PD cells. Flow cytometry and Western Blot were used to detect the effect of ID2-AS1 inhibition on PD cell apoptosis. ELISA analysis showed that ID2-AS1 inhibition can reduce the inflammation of PD cells. ROS activity assay showed that inhibiting ID2-AS1 attenuated the oxidative stress induced by 1-methy1-4-phenylpyridinium (MPP+). RNA binding protein immunoprecipitation assay showed that ID2-AS1 is mainly located in the cytoplasm. The luciferase reporter assay is used to verify the interaction. In our study, ID2-AS1 was concentration-dependently and time-dependently up-regulated in MPP+?-treated human neuroblastoma cell line SH-SY5Y. ID2-AS1 knockdown enhanced cell proliferation and decreased cell death in PD cells. Knockdown of ID2-AS1 attenuates MPP+?-induced cytotoxicity in SH-SY5Y cells. ID2-AS1 is a sponge of miR-199a-5p. IFNAR1 is a target of miR-199a-5p. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+?triggered neuronal injury. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+?-triggered JAK2/STAT1 activation. Overall, down-regulation of ID2-AS1 alleviated the neuronal injury in PD through regulating miR-199a-5p/IFNAR1/JAK2/STAT1 axis.