Cellular Junction Disruption in the Inflammatory Processes and Malignancies - Essay Example

Available evidence has reviewed extensively the available data for developing a rationale to carry out this research work. This literature review has not only been quantitatively but also qualitatively well chosen to support the current research work. Apical Junctional Complex (AJC) represents both the Tight Junctions (TJ) and the Adherens Junction (AJ) two important intercellular connection structures. AJC disassembly is focus for junctional research because of its own practical implications as far as some pathological mechanisms of certain diseases processes including malignancy are concerned. For the explanation of AJC disassembly two more frequently referred mechanisms have been: 1) reorganization of perijunctional cytoskeleton; 2) endocytosis of junctional proteins. Again, these mechanisms need further exploration but the existing understanding is that reorganization of F-actin causes destabilization of transinteraction between TJ and AJ proteins of the adjacent epithelial cells and in turn initiate AJC internalization. Microtubules, which are also an important element of intracellular cytoskeleton have also been found to be closely related to the cell movement as a whole or any intracellular structure formation like, vesiscles and their movement. Data support the abundance of microtubules in differentiated renal and intestinal cells and depolymerization of microtubules leading to disruption of the integrity of TJs and Ajs in some tissues like lungs. This evidence led the author to explore the role of microtubules depolymerization in the disruptions of apical junction complex. Hypothesis of the study Existing evidence strongly support the role of microtubules depolymerization in the disruption of intercellular connections like tight junctions and adherens junction in various tissues and helps in the pathogenesis of various disease processes. In this regard it has been shown that the endothelial intercellular junctions are also disassembled as well as others like in the brain. Building a rationale on the basis of these data, author formulated a hypothesis: 'microtubules are involved in the disruption of apical junctions in simple epithelial cells'. Under this main hypothesis there were sub-hypotheses: 'Disassembly of the AJC in calcium depleted SK-CO-15 cells is dependent on microtubule integrity;' 'Inhibition or attenuation of AJC disassembly by microtubule stabilization would indicate the role for dynamic microtubules in the process;' 'Polarity of perijunctional microtubules would dictate the type of microtubule motor which is involved in disassembly and internalization of the AJC.' To test these hypotheses, the author carried out a research study where a number of experimental activities were carried out, like: use of antibodies for the detection of intercellular junctions, while microtubules were detected by immunoflurescence labeling and Western Blotting. Cell culture was used was SK-CO-15, which is a transformed human colonic epithelial cell line. To make the medium free from Ca++, the monolayers of epithelium were washed with calcium free-Eagle's minimum essential medium for suspension culture. Pharmacological agents utilized were; nocodazole; docetaxel and pacitaxel; adenylimidodiphosphate (AMP-PNP) and aurintriccarboxyylic acid (ATA). Major findings Through this experiment, the author has been able to demonstrate the role of microtubules depolymerization in the disassembly of junctional complexes through a variety of experimental evidence. Disassembly of the AJC in calcium depleted SK-CO-15 cells To answer the research question related to first sub-hypothesis, the findings have been: At normal Ca++ concentrations, depolymerized microtubules with nocodazole TJ and AJ proteins were found to be located at the cell-cell contacts, reflecting chicken-wire staining pattern while in the environment with depletion of Ca++ for one hour the result was disruption of AJs and TJs with accumulation of e-cadherin, occluding and ZO-1 in subapical ring like structures. When the human cells were treated with nocodazole in high concentrations, 30 M, the result was eliminatio nof the apical microtubules, prevention of translocation of all AJ and TJ proteins from cellular junctions to cytosolic ring in the subapical region. The experiment further explored the disassembly that it was not specific to SK-CO-15 cells, nocodazole effect was observed on intestinal and renal cells as well with one hour of Ca++ depletion; the result was similar to as it was with SK-CO-15 cells proving that microtubule dependence of AJC disassembly is universal phenomenon for simple epithelial cells. Attenuation of AJC disassembly by microtubule stabilization There are two types of microtubules, dynamic and stable. Redistribution of dynamic tubules is controlled by polymerization and depolymerization at opposite ends of the filaments while translocation/reorientation of stable microtubules can be driven by microtubule motors. Through this experiment, the author observed the microtubule turnover with relation to the formation of F-actin rings and disassembly of eh AJC in the Ca++ depleted environment. The same human cells, SK-CO-15 cells were first treated with microtubule stabilizing drugs, docetaxel and pacitaxel, followed by one hour of calcium depletion in the presence of the drugs at the same concentrations. The result was high concentration of apical microtubules which proved the role of the these drugs in the stabilization of microtubules while in the depletion of the Ca++, these drugs inhibited the disassembly of AJC and translocation of E-cadherin and occludin from cell-cell contacts in ring like structures. Moreover, these drugs also hampered the formation of apical contractile F-actin rings. So it proved that dynamic microtubules regulte disassembly and internalization of TJs and AJs as well as contraction of perijunctional F-actin microfilaments in Ca++ depleted epithelial cells. Polarity of perijunctional microtubules and the type of microtubule motor in disassembly and internalization of the AJC There are two types of microtubule motors, plus ends and minus ends, depending upon the direction of movement of cargo to these poles. Kinesin proten family are plus end while dynein are minus end. The orientationo f the microtubules in polarized epithelial cells is in a way that the minus ends are towards apex close to apical junction; so the internalization of junctional proteins will result in the movement of these proteins toward plus ends and hence will be mediated by proteins representing plus end, kinesin motors. The author utilized the pharmacological inhibitors of kinesins , adenylylimidodiphosphate (AMP-PNP) and aurintricarboxylic acid (ATA), on AJC disassembly in the environment of calcium depletion on the SK-CO-15 cells. As a result translocation of E-cadherin and occludin from apical junctions to the subapical cytosolic compartment was attenuated by AMP-PNP and ATA, and which in turn blocked contraction of F-actin rings. There are more than 40 kinesin genes in mammals the two most probable candidates are kinesin-1 and kinesin-2. Kinesin-1 is reported to help in the process of trafficking of the AJ proteins N-cadherin and p120 catenin while kinesin-2, on has been reported to interact with the PAR-3-PAR-6-atypical PKC polarity complex in neurons and has been implicated in the formation of the apical plasma membrane domain in polarized kidney epithelial cells. Kinesin-1 appeared to be selectively enriched at the AJC in polarized SK-CO-15 and T84 cells where it colocalized with occludin and -catenin. In calcium-depleted epithelial cells, kinesin-1 redistributed from areas of cell-cell contacts along with junctional proteins and colocalized with internalized occludin and -catenin in the subapical cytosolic compartment. Kinesin-2 staining was dot-like throughout the cell and did not colocalize with AJC proteins in control and calcium-depleted T84 and SK-CO-15 cells. These data suggest that the kinesin-1 motor may play a role in regulating endocytosis and intracellular trafficking of TJ/AJ proteins during calcium depletion. Future implications The information provided by these experiments have been very important as far as the advancement in the cellular morphology and physiology is concerned which may in turn add to the pathological mechanisms of diseases process. As it will be helpful I understanding pathological mechanism, it will also be helpful in the development of pharmacological agents required and thus in the management of the diseases. Specifically, these findings will be helpful in the cells in blood vessels, like endothelial cells especially in the confining the inflammatory process or spread of malignant tumor if the disruption of the intercellular junctions is well understood. All these experiments were performed in the Ca++ depleted scenario and these findings may be of specific interest in the excitable tissues especially cardiac muscle, which is highly dependent on the extracellular concentration of Ca++. Therefore, further research in the areas of cellular junction disruption in the inflammatory processes and malignancies, or the issues of cellular integrity in the presence of Ca++ concentrations at various levels. Read More