Over-Expression Of Caveolin-1 Aggravate LPS-Induced Inflammatory Response In AT-1 Cells And C57BL6 Mice Via Up-Regulation Of CPLA2/p38 MAPK

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are well defined and readily recognized as clinical critical disorders which commonly induced by severe infection, trauma and shock directly and indirectly. The major clinical manifestations include acute respiratory distress, refractory hypoxemia and non-cardiac source pulmonary edema. Mortality rates of acute lung injury is about 30-40%. At present, the pathogenesis of ALI/ARDS has not been fully clarified. Pathological studies have shown that when the diffuse alveolar damage occurs in the ALI/ARDS, the most obvious structure change is the alveolar epithelium, especially typeⅠalveolar epithelial cells, which is more sensitive to injury than typeⅡepithelial cells and the damage often occurs in the early stage.Caveolins are the chief structural proteins of caveolae, which are vesicular invaginations of the plasma membrane. Molecular cloning has identified three distinct caveolin genes, caveolin-1(Cln-1), caveolin-2(Cln-2), and caveolin-3(Cln-3). Cln-1, 22kD, appears to have principal functions in lipid transport, membrane traffic, and cell signaling. There is evidence that Cln-1 is widely present in typeⅠalveolar epithelial cell membrane and that Cln-1/Cle may play an important role in the function of the alveolar epithelial barrier.In the present study, we increased specifically and transiently elevated caveolin-1 expression in AT-1 cells using gene clone technique and studied the effects of over-expression of caveolin-1 on cPLA2/p38 MAPK and the consequent role in the AT-1 cells and the mouse lung injury and in regulating pro-inflammatory cytokines synthesis, nuclear transcription factor activation following LPS challenge.Methods1. The full length cDNA of mouse Cln-1 was cloned by PCR and constructed into lentiviral expression vector pRNAi-u2.6. Recombinant lentivirus vector were packaged using a four-plasmid transient transfection procedure.2. Immunoselection was undertaken to isolate and culture AT-1 cells by incubating cells with the PE-labeled AT-1 cell specific monoclonal antibody (MAb) RTI40. Cell purity was determined by Flow Cytometry.3. The recombinant pRNAi-u2.6-Cln-1 was transfected into AT-1 cells by lentiviral vector. The green fluorescence protein expression was confirmed by inverted fluorescence microscope and the proportion of cells expressing GFP was determined by Flow Cytometry. The expression of Cln-1 were identified by Western blotting.4. AT-1 cells, either transfected with lentiviral vector as experiment group or empty vector (only encoding GFP) as control group, were treated with LPS at concentration 10μg/ml at 2hrs and 4hrs. The level of cytokines (TNF-α, IL-6) released by LPS-stimulated AT-1 cells into the culture supernatant were measured by ELISA kits. The mRNA expression of cPLA2 and p38 MAPK were measured by real-time PCR. Western blotting was used to verify the result of real-time PCR. The expression of NF-κB was detected by EMSA. MAFP (the inhibitor of cPLA2), SB203580 (the inhibitor of p38 MAPK) and BAY11-7082 (the inhibitor of NF-κB) were added to examine the effect on the production of cytokines (TNF-α, IL-6) and the inter-action among cPLA2, p38 MAPK, and NF-κB.5. c57BL6 mice, injected of caveolin-1 lentiviral vector via tail vein, were treated with LPS at concentration 2mg/kg at 2hrs and 4hrs one month later. The level of cytokines (TNF-α, IL-6) in serum were measured by ELISA kits. The expression of cPLA2 and p38 MAPK were detected by Western blotting and lung histological sections were analyzed.Results1. The Cln-1 gene full length cDNA was amplified by PCR and cloned into pRNAi-u2.6, and sequencing result showed that the sequence of Cln-1 cDNA was in coincidence with the sequence in Genbank. Lentiviral transfer vector encoding the GFP marker gene was packaged by a standard four-plasmid cotransfection procedure. The titers of concentrated vector supernatants generated by this procedure are typically on the order of 2.5×10~8 TU/ml.2. Type I alveolar epithelial cells were obtained. The purity of type I cells ranged from 85% to 98%. Yields were 3-5×10~6 cells. 3. Lentiviral vector was transfected into AT-1 cells. The green fluorescence protein was confirmed by inverted fluorescence microscope. Representative FACS analysis at 3 days post-transfection shows a shifted population of cells exhibiting higher fluorescence intensity specifically in the GFP wavelength (FL1 channel), with >95% GFP-positive cells, demonstrating that AT-1 cells are efficiently transduced by this vector. At 36hr post-transfection, caveolin-1 protein levels were significantly increased with the peak value at 72hrs compared with that of the control, as evaluated by Western blotting analysis.4. The over-expression of caveolin-1 caused a significant increase in pro-inflammatory cytokines TNF-αand IL-6 at 2hr and 4hr. The mRNA expression of cPLA2 and p38 MAPK were significantly increased in caveolin-1 over-expressing cells at 2hrs and 4hrs induced by LPS. The activation of the total and phosphorylated cPLA2 and p38 MAPK were significantly increased correspondingly. In the experiment group, the expression of NF-κB significantly increased as comparison to that in control group. MAFP (the inhibitor of cPLA2), SB203580 (the inhibitor of p38 MAPK) and BAY11-7082 (the inhibitor of NF-κB) decreased TNF-αand IL-6 production compared with DMSO/LPS treatment. Pre-treatment with MAFP reduced the activation of phosphorylated p38 MAPK and NF-κB, compared with vector control (DMSO). When the SB203580 was administered to AT-1 cells, the phosphorylated cPLA2 and NF-κB were inhibited, whereas the phosphorylated cPLA2 and p38 MAPK were not affected when BAY11-7082 was added.5. The expression of pro-inflammatory cytokines (TNF-αand IL-6) in the serum of over-expression of caveolin-1 mice increased significantly than that in the control induced by LPS. So did as to the expression of cPLA2 and p38 MAPK according to the change in the AT-1 cells. The analysis of lung tissue pathology showed that the pulmonary effusion, edema is more evident, and the damage is more serious in the experiment group than that in the control.Conclusion1. The model of over-expression caveolin-1 in the AT-1 and c57BL6 cells was successfully constructed.2. The over-expression of caveolin-1 aggravates injury in the AT-1 cells by up-regulation of pro-inflammatory cytokine (TNF-αand IL-6) production. We speculate that the increased activation of cPLA2 and p38 MAPK and downstream molecule lead to inflammation aggravation.3. The specific blocking of cPLA2, p38 MAPK and NF-κB can reduce inflammatory response induced by LPS.