Down-regulation of HIV-1 Infection by Inhibition of the MAPK Signaling Pathway

The human immunodeficiency virus type 1 （HIV-1） can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase （MAPK） signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase （ERK） pathway inhibitor, PD98059, the Jun N-terminal kinase （JNK） pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1NL4-3 luciferase reporter virus and HIV-1NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-INL4-3 luciferase reporter virus inhibition activity but no HIV-1NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.

Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

光线性角化病和鳞状细胞癌表皮生长因子受体与MAPK信号通路的研究

目的探讨磷酸化ERKl／2、JNK和p38MAPK与其上游因子表皮生长因子受体（EGFR）及下游转录因子Elk-1在光线性角化病（AK）和皮肤鳞状细胞癌（scc）中的表达。方法收集确诊为AK和高、中、低分化SCC患者各30例的病变组织，同时收集30例非癌患者正常皮肤组织作为正常对照组。采用免疫组化及Western印迹法分别检测p-EGFR、P—ERKI~、P—JNK、P—p38MAPK及p—Elk-1蛋白在AK和不同分化程度SCC中的表达情况。结果P—EGFR在SCC组中的表达高于AK组及正常对照组（P〈0．05），AK组与正常对照组表达差异无统计学意义；P—ERKl／2、P—JNK、P—p38MAPK及p-Elk-1在SCC的表达均高于AK组及正常对照组（P〈0．05），且在AK中的表达又均高于正常对照组（P〈0．05）。除p-EGFR和p-Elk-1在高分化和中分化SCC中的表达差异无统计学意义，p-ERKl／2、P—JNK、P—p38MAPK随着SCC分化程度的降低，其表达逐渐增高（P〈0．05）。相关性分析显示，在SCC中p-EGFR的表达与p-ERKI~、P—JNK、P—p38MAPK的表达强度呈正相关（r=0．843、0．819、0．902，均P〈0．01），且p-Elk-1的表达与p-ERKl／2、P—JNK、P—p38MAPK的表达强度亦呈正相关（r=0．874、0．843、0．893，均P〈0．01）；在AK中P—EGFR的表达仅与P—p38MAPK的表达强度呈正相关（r=0．707，P=0．022）。结论p-EGFR、P—ERKI、P—JNK、P—p38MAPK及P—Elk-1蛋白在皮肤SCC中的表达与其病理分级有关。磷酸化EGFR--ERKI／2、JNK、p38MAPK---Elk-1信号转导途径可能在AK和SCC的发生发展中起作用。