The Role And Mechanisms Of MAP4 In Inflammation-induced Lung Microvascular Hyperpermeability

BackgroundSevere burns lead to local or systematic activation of inflammation and the accompanying production of many inflammatory mediators, which can directly or indirectly cause damage of endothelial cells and hyperpermeability. Lung as a bridge to the environment, it is easily affected by many factors, which lead to increased lung microvascular permeability and acute lung injury(ALI). However, there are still no effective ways to decrease hyperpermeability, and the underlying regulated mechanisms of inflammation-induced lung microvascular hyperpermeability still unclear.Recently, increasing data have suggested that microtubule(MT) is essential for the integrity of endothelial barrier, and the dynamics of microtubule has associated with many factors, such as GTP, temperature, drugs and microtubule associated proteins(MAPs). Some data indicated that MT disassembly lead to injury of cell morphology and cell-cell junctional structures, which can be prevented by application of microtubule stabilizer. However, the detailed regulated mechanisms of microtubule in inflammation-induced endothelial barrier dysfunction still unknown.As a well-known tubulin associated protein, microtubule-associated protein 4(MAP4) has play an important role in the maintanence of structures and dynamics of microtubule. Once it is phosphorylated, MAP4 dissociates from microtubule, resulting in MT instability. We previously confirmed that p38/MAPK pathway activation lead to MAP4 phosphorylation with microtubule disassembly in caridomyocytes during hypoxia; and p38/MAPK is an important signal pathway required for microfilament rearrangement that precedes endothelial barrier dysfunction. However, the involvement and the underlying regulated mechanisms of MAP4 in mediating inflammation-induced endothelial barrier dysfunction in ALI remains unknown.Thus, we would first determine that whether MAP4(S696, S787, S768) phosphorylation increased in inflammation. Then we would construct a MAP4(Ala) mutant, which mimicked the dephosphorylated form, by changing Ser696, Ser787, and Ser768 to alanine, to demonstrate that whether MAP4 phosphorylation was important in mediating inflammation-induced lung microvascular hyperpermeability. On this basis, we would also elucidate the underlying regulated mechanisms of MAP4 in inflammation-induced endothelial barrier dysfunction in ALI.Materials and Methods1. To study the effect of inflammatory treatment on endothelial barrier permeability and MTs, we applied LPS(200, 500 and 1000ng/ml) and TNF-α(200, 500 and 1000ng/ml) to stimulate human pulmonary microvascular endothelial cells(HPMECs) in vitro. The permeability was assessed by measuring the leakage of FITC-dextran and transendothelial electrical resistance(TER) across monolayer cells. The MT structures were showed by immunofluorescence(IF), and the MT fractions were analyzed by western blot(WB).2. To elucidate whether MT disassembly involve in inflammation-induced endothelial permeability, the HPMECs were pretreated with or without taxol(1uM) under LPS(500ng/ml) or TNF-α(500ng/ml) stimulation. The permeability was assessed by measuring the leakage of FITC-dextran and TER across monolayer cells. The MT structures were showed by IF, and the MT fractions were analyzed by WB.3. To determine whether LPS and TNF-α induce MAP4 phosphorylation, HPMECs were treated with or without LPS(500ng/ml) or TNF-α(500ng/ml) at indicated time and analyzed by WB; we next constructed a MAP4(Ala) mutant to mimick the dephosphorylated form of MAP4, and then determined the effect of MAP4 phosphorylation on endothelial barrier permeability and MTs, cells were transfected with or without MAP4(Ala) in inflammation. The permeability was assessed by measuring the leakage of FITC-dextran and TER across monolayer cells. The MTs were showed by IF and WB, and the combination of MAP4 with microtubule was analyzed by Immunoprecipitation(IP).4. To address whether p38/MAPK mediates MAP4 phosphorylation in inflammation, HPMECs were treated with or without LPS(500ng/ml) or TNF-α(500ng/ml) at indicated time and analyzed by WB; and then we constructed MKK6(Glu) to activate the p38/MAPK pathway. In contrast, p38/MAPK inhibitor SB203580(5u M) was used to inhibit the p38/MAPK activation under the inflammatory stimulation, MAP4(S696, S787 and S768) and p38/MAPK phosphorylation was measured by WB and the combination of MAP4 with tubulin were assessed by IP.5. To observe the effect of p38/MAPK on endothelial barrier permeability and MTs, SB203580(5uM) and MKK6(Glu) were applied with or without LPS(500ng/ml) or TNF-α(500ng/ml) stimulation. Then we addressd the role of MAP4 phosphorylation in mediating the p38/MAPK regulation of endothelial barrier permeability and MT dynamics, the cells were transfected or cotransfected with CMV-null, MAP4(Ala) or MKK6(Glu). The permeability was assessed by measuring the leakage of FITC-dextran and TER across monolayer cells. The MT structures were showed by IF, and the MT fractions were analyzed by WB.6. To confirm that MAP4 phosphorylation increased the endothelial barrier permeability in a MT-dependent manner independent of apoptosis, caspase-3 inhibitor(Z-DQMD-FMK, 10 u M), MAP4(Ala) or SB203580(5u M) were applied before LPS(500ng/ml) or TNF-α(500ng/ml) stimulation. The apoptosis was measured by TUNEL and the permeability was assessed by measuring the leakage of FITC-dextran and TER across monolayer cells.Results1. We found that LPS and TNF-α induced endothelial hyperpermeability, as indicated by increased FITC-dextran leakage and reduced TER in a dose-dependent manner. Morphologically, LPS(200ng/ml) and TNF-α(200ng/ml) caused some breakages and a less regular organization. The disruption continued with the increase in LPS(500ng/ml) and TNF-α(500ng/ml), there was obvious MT disruptions along the cell membrane and some shrinkage near the nuclei. With further LPS(1000ng/ml) and TNF-α(1000ng/ml) stimulation, only a thin and disorderd residual MT organization and fractions remained. Besides, we demonstrated a concentration-dependent increase in free tubulin with a parallel decrease in polymerized tubulin.2. The results suggested that taxol(1uM) pretreatment attenuated the LPS(500ng/ml) or TNF-α(500ng/ml)-induced increased endothelial cell permeability, as represented by the FITC-dextran leakage and the TER. The taxol pretreatment also led to protection of the MT organization and showed an increased polymerized tubulin as well as decreased free tubulin.3. We found that LPS(500ng/ml) and TNF-α(500ng/ml) treatment induced MAP4(S696 and S787) phosphorylation in a time-dependent manner but not at MAP4(S768). Then we transfected MAP4(Ala) into HPMECs, the results indicated that MAP4(Ala) could decrease FITC-dextran, increase TER, and protect against MT disassembly in inflammation. Besides, MAP4(Ala) promoted MAP4 interact with tubulin.4. We investigated that LPS(500ng/ml) and TNF-α(500ng/ml) activated p38/MAPK in a time dependent manner in inflammation. Then, MKK6(Glu) was transfected into HPMECs to activate p38/MAPK, the results suggested that MKK6(Glu) induced MAP4(S696 and S787) and p38/MAPK phosphorylation and showed the dissociation of tubulin with MAP4. In contrast, SB203580(5u M) pretreatment inhibited MAP4(S696 and S787) phosphorylation and p38/MAPK activation under LPS(500ng/ml) or TNF-α(500ng/ml) treatment, and protected against dissociation of tubulin with MAP4 in inflammation.5. We found that SB203580(5uM) largely abolished the LPS(500ng/ml) and TNF-α(500ng/ml)-induced hyperpermeability and MT disassembly. While transduction with MKK6(Glu) resulted in hyperpermeability and MT disruption, and led to an increase in free tubulin and decrease in polymerized tubulin. Then we transfected or cotransfected with CMV-null, MAP4(Ala) or MKK6(Glu), we observed that MAP4(Ala) prevented the p38/MAPK activation of hyperpermeability and were more resistant than CMV-null to MT disassembly in response to MKK6(Glu) transfection.6. HPMECs were pretreated with caspase-3 inhibitor(Z-DQMD-FMK, 10uM) to block the LPS(500ng/ml) or TNF-α(500ng/ml)-induced cell apoptosis. The results indicated that pretreatment with caspase-3 inhibitor(10uM), SB203580(5u M) or MAP4(Ala) pretreatment showed a more protective effect against hyperpermeability under LPS(500ng/ml) or TNF-α(500ng/ml) stimulation.ConclusionsWe confirmed that the MAP4(S696 and S787) phosphorylation increased concomitantly with the p38/MAPK activation by the LPS or TNF-α treatment of HPMECs, which induced microtubule disassembly followed by lung microvascular hyperpermeability, and the MAP4 phosphorylation increased the lung microvascular permeability develops independently of apoptosis. Our data illustrate a novel function of MAP4 in endothelial barrier dysfunction and imply a possible therapeutic strategy targeting MAP4 phosphorylation against vascular hyperpermeability in ALI.