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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. Na-K-ATPase [[alpha].sub.1]-subunit, Na-K-ATPase [[beta.sub.1]-subunit; protein phosphatase-2A; pancreas

Na-K-ATPase, also known as the sodium pump, is a crucial enzyme that regulates intracellular sodium homeostasis in mammalian cells. In epithelial cells Na-K-ATPase function is also involved in the formation of tight junctions through RhoA GTPase and stress fibers. In this review, a new two-step model for the assembly of tight junctions is proposed: step 1, an E-cadherin-dependent formation of partial tight junction strands and of the circumferential actin ring; and step 2, active actin polymerization-dependent tethering of tight junction strands to form functional tight junctions, an event requiring normal function of Na-K-ATPase in epithelial cells. A new role for stress fibers in the assembly of tight junctions is proposed. Also, implications of Na-K-ATPase function on tight junction assembly in diseases such as cancer, ischemia, hypomagnesemia, and polycystic kidney disease are discussed. E-cadherin; stress fibers; ischemia

Na,K-ATPase regulates a variety of transport functions in epithelial cells. In cultures of human retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPase by ouabain and [K.sup.+] depletion decreased transepithelial electrical resistance (TER) and increased permeability of tight junctions to mannitol and inulin. Electrophysiological studies demonstrated that the decrease in TER was due to an increase in paracellular shunt conductance. At the light microscopy level, this increased permeability was not accompanied by changes in the localization of the tight junction proteins ZO-1, occludin, and claudin-3. At the ultrastructural level, increased tight junction permeability correlated with a decrease in tight junction membrane contact points. Decreased tight junction membrane contact points and increased tight junction permeability were reversible in [K.sup.+]-repletion experiments. Confocal microscopy revealed that in control cells, Na,K-ATPase was localized at both apical and basolateral plasma membranes. [K.sup.+] depletion resulted in a large reduction of apical Na,K-ATPase, and after [K.sup.+] repletion the apical Na,K-ATPase recovered to control levels. These results suggest a functional link exists between Na,KATPase and tight junction function in human RPE cells. actin stress fibers

The activities of Tsk and Btk, members of the pleckstrin-homology (PH) domain containing tyrosine kinase family, are increased by some beta and gamma subunits of guanine nucleotide-binding proteins (G proteins). The PH region can react with the beta-gamma subunits of G proteins and phospholipids. Although the exact function of the PH domain is unclear, it is presumed to play a role in protein-protein interactions.

Sucrose-density gradient analysis and cell fractionation techniques help isolate and quantify cultured cell- and solid tissue-derived membrane-bound ribosomes, which enables a determination of the amount of rough endoplasmic reticulum (RER) in these cells. Cells active in secretion possess more RER, as revealed by a measurement of the ratio between free and membrane-bound ribosomes in secretory and nonsecretory cell lines.

Abstract Background Medulloblastoma is the most common brain tumor in children, and its prognosis is worse than for many other common pediatric cancers. Survivors undergoing treatment suffer from serious therapy-related side effects. Thus, it is imperative to identify safer, effective treatments for medulloblastoma. In this study we evaluated the anti-cancer potential of curcumin in medulloblastoma by testing its ability to induce apoptosis and inhibit tumor growth in vitro and in vivo using established medulloblastoma models. Methods Using cultured medulloblastoma cells, tumor xenografts, and the Smo/Smo transgenic medulloblastoma mouse model, the antitumor effects of curcumin were tested in vitro and in vivo. Results Curcumin induced apoptosis and cell cycle arrest at the G2/M phase in medulloblastoma cells. These effects were accompanied by reduced histone deacetylase (HDAC) 4 expression and activity and increased tubulin acetylation, ultimately leading to mitotic catastrophe. In in vivo medulloblastoma xenografts, curcumin reduced tumor growth and significantly increased survival in the Smo/Smo transgenic medulloblastoma mouse model. Conclusions The in vitro and in vivo data suggest that curcumin has the potential to be developed as a therapeutic agent for medulloblastoma.

Purpose: Na,K-adenosine triphosphatase, which is composed of a catalytic α-subunit and a regulatory β-subunit, generates an electrochemical gradient across the plasma membrane. Previous studies demonstrated altered Na,K-adenosine triphosphatase subunit expression in renal clear cell carcinoma and an association of subunit levels with the prediction of recurrent bladder cancer. We determined the clinical association of protein expression patterns of the Na,K-adenosine triphosphatase α1 and β1-subunits in renal clear cell carcinoma using tissue microarrays with linked clinicopathological data. Materials and Methods: The UCLA kidney cancer tissue microarray was used to investigate the protein expression of Na,K-adenosine triphosphatase α1 and β1-subunits by immunohistochemistry in 342 patients with renal clear cell carcinoma who were treated with radical nephrectomy. Of these patients clinical outcomes studies were performed in 317. The resultant expression reactivity was correlated with clinicopathological variables. Results: We found that the α1-subunit was a significant and independent predictor of disease specific death from renal clear cell carcinoma on multivariate Cox proportional hazards analysis that included established prognostic factors Eastern Cooperative Oncology Group performance status, pT status, metastasis status and tumor grade. Significance was found when examining all patients with clear cell renal cell carcinoma as well as patient substrata with low or high grade tumors and localized or metastatic disease, suggesting that the Na,K-adenosine triphosphatase α1-subunit could be used as a new prognosticator for disease specific death from renal clear cell carcinoma. Conclusions: These results suggest that Na,K-adenosine triphosphatase α1-subunit expression patterns may be a useful clinical prognosticator for renal clear cell carcinoma. The Na,K-adenosine triphosphatase β1-subunit was not found to be a useful prognosticator in this setting. [Copyright &y& Elsevier]