Biomedical subjects

Ayyappan K Rajasekaran

Publications and source records attributed to Ayyappan K Rajasekaran.

At least 19 recordsLinked to original sources

PubMed2006

Janus model of the Na,K-ATPase beta-subunit transmembrane domain: distinct faces mediate alpha/beta assembly and beta-beta homo-oligomerization.

Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.

Amino Acid Sequence↗

PubMed2006

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells.

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.

Cadherins↗

PubMed2006

Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis.

For most carcinomas, progression toward malignancy is accompanied by loss of epithelial differentiation and a shift towards a mesenchymal phenotype. This process, referred to as epithelial to mesenchymal transition (EMT), exacerbates motility and invasiveness of many cell types and is often considered a prerequisite for tumor infiltration and metastasis. However, there are numerous examples of advanced carcinomas that adopt some mesenchymal features, yet retain characteristics of well-differentiated epithelial cells. We provide a review of these reports and describe mechanisms to explain the morphologic and molecular heterogeneity and plasticity of malignant carcinoma cells, including incomplete EMT, reversion to an epithelial phenotype, and collective migration. We suggest that these mechanisms can manifest in a series of independent and reversible steps and that EMT represents just one mechanism in the global metastatic carcinoma development process.

Carcinoma↗

PubMed2006

Association of prostate-specific membrane antigen with caveolin-1 and its caveolae-dependent internalization in microvascular endothelial cells: implications for targeting to tumor vasculature.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein with a highly restricted profile of expression. Expression is primarily limited to secretory cells of the prostatic epithelium, with elevated levels observed in prostate cancer. As an integral membrane protein correlated with prostate cancer, PSMA offers a potentially valuable target for immunotherapy. PSMA is also detected in the neovasculature of a variety of solid tumors but not in the endothelial cells of preexisting blood vessels. Although the significance of PSMA expression in these cells remains elusive, this pattern of expression implies that PSMA may perform a functional role in angiogenesis and may offer a therapeutic target for the treatment of a broad spectrum of solid tumors. In this study, we have expressed PSMA in human microvascular endothelial cells and demonstrate that PSMA binds to caveolin-1 and undergoes internalization via a caveolae-dependent mechanism. The association between PSMA and caveolae in endothelial cells may provide important insight into PSMA function and ways to best exploit this protein for therapeutic benefit.

Antigens, Surface↗

PubMed2006

Estrogen receptor-alpha binds p53 tumor suppressor protein directly and represses its function.

Estrogen receptor-alpha (ERalpha) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERalpha and p53. Ionizing radiation together with ERalpha knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the pro-proliferative role of ERalpha.

Breast Neoplasms↗

PubMed2006

Differing effects of microtubule depolymerizing and stabilizing chemotherapeutic agents on t-SNARE-mediated apical targeting of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.

Animals↗

PubMed2005

Multiple functions of Na,K-ATPase in epithelial cells.

The Na,K-adenosine triphosphatase (ATPase), or sodium pump, has been well studied for its role in the regulation of ion homeostasis in mammalian cells. Recent studies suggest that Na,K-ATPase might have multiple functions such as a role in the regulation of tight junction structure and function, induction of polarity, regulation of actin dynamics, control of cell movement, and cell signaling. These functions appear to be modulated by Na,K-ATPase enzyme activity as well as protein-protein interactions of the alpha and beta subunits. In this review we attempt to differentiate functions associated with enzyme activity and subunit interactions. In addition, the consequence of impaired Na,K-ATPase function or reduced subunit expression levels in kidney diseases such as cancer, tubulointerstitial fibrosis, and ischemic nephropathy are discussed.

Animals↗

PubMed2005

Is prostate-specific membrane antigen a multifunctional protein?

Prostate-specific membrane antigen (PSMA) is a metallopeptidase expressed predominantly in prostate cancer (PCa) cells. PSMA is considered a biomarker for PCa and is under intense investigation for use as an imaging and therapeutic target. Although the clinical utility of PSMA in the detection and treatment of PCa is evident and is being pursued, very little is known about its basic biological function in PCa cells. The purpose of this review is to highlight the possibility that PSMA might be a multifunctional protein. We suggest that PSMA may function as a receptor internalizing a putative ligand, an enzyme playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into the possible functions of PSMA should improve the diagnostic and therapeutic values of this clinically important molecule.

Antigens, Neoplasm↗

PubMed2005

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy.

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.

Animals↗

PubMed2004

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility.

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.

Actins↗

PubMed2004

Na,K-ATPase beta1-subunit increases the translation efficiency of the alpha1-subunit in MSV-MDCK cells.

The Na,K-ATPase consists of an alpha- and beta-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase beta(1)-subunit. Ectopic expression of Na,K-ATPase beta(1)-subunit in these cells increased the protein levels of the alpha(1)-subunit of Na,K-ATPase. This increase was not due to altered transcription of the alpha(1)-subunit gene or half-life of the alpha(1)-subunit protein because both alpha(1)-subunit mRNA levels and half-life of the alpha(1)-subunit protein were comparable in MSV-MDCK and beta(1)-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the alpha(1)-subunit in beta(1)-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to alpha(1)-subunit because translation rates of occludin and beta-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the alpha(1)-subunit mRNA is more efficiently translated in the presence of beta(1)-subunit. Furthermore, sucrose density gradient analysis revealed significantly more alpha(1)-subunit transcript associated with the polysomal fraction in beta(1)-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase beta(1)-subunit is involved in facilitating the translation of the alpha(1)-subunit mRNA in the endoplasmic reticulum.

Animals↗

PubMed2004

Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line.

Local invasiveness is a characteristic feature of glioblastoma that makes surgical resection nearly impossible and accounts in large part for its poor prognosis. To identify mechanisms underlying glioblastoma invasion and motility, we used Transwell invasion chambers to select for a more potently invasive subpopulation of U87MG human glioblastoma cells. The stable population of tumor cells (U87-C1) obtained through this in vitro selection process were three times more invasive than parental U87MG cells and demonstrated faster monolayer wound healing and enhanced radial motility from cell spheroids. This enhanced invasiveness was associated with an 80% increase in matrix metalloproteinase 2 (MMP-2) activation. No differences in expression levels of pro-MMP-2, membrane-type matrix metalloproteinase I (MT1-MMP), or integrin alphavbeta3 (mediators of MMP-2 activation) were detected. However, U87-C1 cells exhibited two-fold elevation of tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and protein relative to parental cells. Exogenous addition of comparable levels of purified TIMP-2 to parental U87MG cells increased MMP-2 activation and invasion. Similarly, U87MG cells engineered to overexpress TIMP-2 at the same levels as U87-C1 cells also demonstrated increased MMP-2 activation, indicating that an increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioblastoma cells. However, exogenous administration or recombinant overexpression of higher amounts of TIMP-2 in U87MG cells resulted in inhibition of MMP-2 activation. These results demonstrate that the complex balance between TIMP-2 and MMP-2 is a critical determinant of glioblastoma invasion, and indicate that increasing TIMP-2 in glioblastoma patients may potentially cause adverse effects, particularly in tumors containing high levels of MT1-MMP and MMP-2.

Cell Line, Tumor↗

PubMed2004

HPAF-II, a cell culture model to study pancreatic epithelial cell structure and function.

OBJECTIVES: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas. METHODS: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy. RESULTS: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and beta-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase beta-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells. CONCLUSION: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.

Adherens Junctions↗

PubMed2004

Biological impediments to monoclonal antibody-based cancer immunotherapy.

The ability of antibodies to exploit antigenic differences between normal and malignant tissues and to exact a variety of antitumor responses offers significant advantages to conventional forms of therapy. Several monoclonal antibodies (mAb) have already proved to be relatively well tolerated and effective for the treatment of many different malignant diseases. However, mAbs must overcome substantial obstacles to reach antigens presented on target cells to be of therapeutic value. Intravenously administered antibodies must avoid host immune response and contend with low or heterogeneous expression of antigen on tumor cells. Antibodies must also overcome significant physical barriers en route to a solid tumor mass, including the vascular endothelium, stromal barriers, high interstitial pressure, and epithelial barriers. Here we review the application and evolution of mAbs as effective forms of treatment, with particular attention to the barriers and impediments to successful treatment and discuss strategies to overcome these barriers and improve the efficacy of mAb-based therapy.

Animals↗

PubMed2003

Repression of Na,K-ATPase beta1-subunit by the transcription factor snail in carcinoma.

The Na,K-ATPase consists of two essential alpha- and beta-subunits and regulates the intracellular Na+ and K+ homeostasis. Although the alpha-subunit contains the catalytic activity, it is not active without functional beta-subunit. Here, we report that poorly differentiated carcinoma cell lines derived from colon, breast, kidney, and pancreas show reduced expression of the Na,K-ATPase beta1-subunit. Decreased expression of beta1-subunit in poorly differentiated carcinoma cell lines correlated with increased expression of the transcription factor Snail known to down-regulate E-cadherin. Ectopic expression of Snail in well-differentiated epithelial cell lines reduced the protein levels of E-cadherin and beta1-subunit and induced a mesenchymal phenotype. Reduction of Snail expression in a poorly differentiated carcinoma cell line by RNA interference increased the levels of Na,K-ATPase beta1-subunit. Furthermore, Snail binds to a noncanonical E-box in the Na,K-ATPase beta1-subunit promoter and suppresses its promoter activity. These results suggest that down-regulation of Na,K-ATPase beta1-subunit and E-cadherin by Snail are associated with events leading to epithelial to mesenchymal transition.

Animals↗

PubMed2003

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

Adaptor Protein Complex 2↗

PubMed2003

Prostate-specific membrane antigen association with filamin A modulates its internalization and NAALADase activity.

Prostate-specific membrane antigen (PMSA) is an integral membrane protein highly expressed by prostate cancer cells. We reported previously that PSMA undergoes internalization via clathrin-coated pits (Liu et al., Cancer Res., 58: 4055-4060, 1998). In this study we demonstrate that filamin A, an actin cross-linking protein, associates with the cytoplasmic tail of PSMA and that this association of PSMA with filamin is involved in its localization to the recycling endosomal compartment. By ectopically expressing PSMA in filamin-negative and -positive cell lines, we additionally show that filamin binding to PSMA reduces the internalization rate of PSMA and its N-acelylated-alpha linked-acidic dipeptidase activity. These results suggest that filamin might be an important regulator of PSMA function.

Amino Acid Sequence↗

PubMed2003

Analysis of the Na,K-ATPase alpha- and beta-subunit expression profiles of bladder cancer using tissue microarrays.

BACKGROUND: The purpose of this study was to determine the clinical significance of Na,K-ATPase alpha- and beta-subunit expression in a histopathologically well-characterized group of patients representing a wide spectrum of tumor grades and disease stages with transitional cell carcinomas (TCC). METHODS: Na,K-ATPase alpha- and beta-subunit protein expression patterns were analyzed using immunohistochemistry on urothelial cancer tissue microarrays (TMA) of 146 patients diagnosed with urothelial carcinoma. For each subunit, the maximum staining intensity and the percentage of positive cells staining at the maximal intensity were analyzed. RESULTS: Compared with the benign fields, the mean protein expression for both Na,K-ATPase alpha- and beta-subunits were found to be decreased overall in in situ and invasive tumors, as well as in tumor-adjacent dysplastic fields. When Na,K-ATPase alpha- and beta-subunit expression levels were dichotomized into distinct groups, they were both found to be significant predictors of recurrence risk in multivariate logistic regression analysis (P = 0.0062, odds ratio [OR] = 2.6 and P = 0.013, OR = 0.43, for Na,K-ATPase alpha- and beta-subunits, respectively). The authors also found that patients with high alpha- and low beta-subunit expression had a high risk for early recurrence, whereas patients with a low alpha- and high beta-subunit expression had a significantly longer median recurrence-free time (17 months and 125 months, respectively, log rank statistics P = 0.0005). CONCLUSIONS: The results suggested that Na,K-ATPase alpha- and beta-subunit expression levels may be useful predictors of clinical outcomes such as recurrence-free time of bladder cancer patients.

Adult↗