The subcellular compartmentalization of TGFß-RII and the dynamics of endosomal formation during the signaling events: An in vivo study on rat mesothelial cells.

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-ß was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-ß signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-ß signaling. Using immunocytochemical approach, we could detect TßRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TßRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TßRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-ß signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions.

Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells.

The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localisation: an in vivo study.

Recently, it has been shown that 17beta estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the alpha oestrogen receptor (ERalpha) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERalpha interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERalpha is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERalpha. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.

Expression of NTPDase1 and caveolins in human cardiovascular disease.

Pathological circumstances like inflammation or ischemic insult facilitate the release of adenine nucleotides from several types of cells. These extracellular nucleotides are rapidly converted to adenosine by ectonucleotidases, mainly ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) and CD73. NTPDase1/CD39 can interact with caveolins, structural proteins of signal-transducing microdomains termed caveolae. Caveolins are thought to have physiological roles in heart ageing and cardiac diseases. The aim of this study was to investigate the expression of NTPDase1 together with caveolins in chronic human cardiovascular diseases and elucidate their role in human heart. The HPLC analysis showed significant increase in ATPase activity in pathological samples from patients with ischemic heart disease. Immunostaining also showed alterations in the expression and distribution of NTPDase1. Caveolin-1 and caveolin-2 expression was much alike in control and pathological cases, while expression of caveolin-3 was lower in pathological samples. Changes in the expression of NTPDase1 and caveolins seem to be independent of human cardiovascular disease.

Ocadaic acid treatment causes tyrosine phosphorylation of caveolin-2 and induces internalization of caveolae in rat peritoneal macrophages.

In this paper, we provide evidences that protein phosphatases could regulate the internalization cycle of caveolae in rat peritoneal cells. Ocadaic acid (OA)--a serine/threonine phosphatase inhibitor--was used in various concentrations (4 and 100 nM) to study the internalization of horseradish peroxidase (HRP) in resident and elicited macrophages. We have found that OA in both concentrations has significantly decreased HRP uptake in resident and elicited cells. The results of our morphometrical analysis showed that in OA-treated cells, the number of surface-connected caveolae has been dramatically decreased. Simultaneously large, endosome-like vacuoles containing small vesicles appeared in the cytoplasm. The membrane of these small vesicles was labeled with anti-caveolin-1 antibody. Immunoprecipitation and Western blot analysis revealed that in OA-treated cells an approximately 29 kDa protein identified as caveolin-2 in macrophages was phosphorylated on tyrosine residues. These data support the idea that there is a close correlation between the phosphorylation of caveolin-2 and endocytosis of caveolae: the tyrosine phosphorylation of this approximately 29 kDa protein can drive caveolae to pinch off from the plasma membrane and causes accumulation of caveolae in a multivesicular body-like cellular compartment, which was never found to contain lysosomal enzymes. As a result of OA treatment caveolin-2 remains phosphorylated and the phosphorylation of these protein might inhibit the recycling of caveolae.

Utilization of lacrimal urea assay in the monitoring of hemodialysis: conditions, limitations and lacrimal arginase characterization.

The lacrimal urea content was found to be proportional to that of blood, which suggested its possible utilization in the monitoring of hemodialysis as a less invasive method. On the other hand, however, arginase activity was detected in tears, which may influence the urea content independently of blood urea concentration. The feasibility of using lacrimal urea measurement to replace blood urea measurement in the monitoring hemodialysis was also investigated. Blood and tear samples of 35 healthy persons and 43 renal patients undergoing hemodialysis were tested. Tear samples were collected on Schirmer paper strips. After elution the lacrimal urea content was measured by a colorimetric method. The determination of arginase activity was based on the release of urea and ornithine. The correlation between blood and lacrimal urea and arginase was studied by multivariate regression analysis. The lacrimal arginase isoenzyme pattern was investigated by native polyacrylamide gel electrophoresis and Western blotting. The effect of partially isoform-specific inhibitors was also studied. Blood urea levels in blood were significantly higher in the renal patients before dialysis than in the control patients (12.86 +/- 0.59 vs. 6.45 +/- 0.41 mM, p < 0.0001). Blood sera arginase activity was very low. Lacrimal arginase activity was significantly higher in tears than in sera (p < 0.0001 for each group). The tear/serum ratio of urea content was significantly different between controls and renal patients, particularly in postdialytic samples (1.89 +/- 0.07 vs. 3.49 +/- 0.31, p < 0.0001). The correlation between lacrimal and blood sera urea was best in controls (r = 0.89) and was better in predialytic (r = 0.75) than in postdialytic (r = 0.52) samples, depending on the level of arginase activity. In postdialytic samples a stronger correlation (r = 0.77) between tear urea and arginase was observed. Both arginase isoforms were detected in tears, but the extrahepatic (arginase II) isoenzyme was present in higher concentration. In conclusion, the determination of lacrimal urea level as a possible less invasive replacement for blood urea determination could only be utilized in the monitoring of hemodialysis if lacrimal arginase is also measured. Blood urea levels can be correctly determined by using equations, which take into account arginase activity. The accuracy of these equations was checked on a new patient population. Both arginase isoenzymes were observed in lacrimal samples.

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis.

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells). We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.