Limits...
A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling.

Hu M, Wang C, Li W, Lu W, Bai Z, Qin D, Yan Q, Zhu J, Krueger BJ, Renne R, Gao SJ, Lu C - PLoS Pathog. (2015)

Bottom Line: Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion.Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT.Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P. R. China; Key Laboratory Of Pathogen Biology Of Jiangsu Province, Nanjing Medical University, Nanjing, P. R. China; Department of Microbiology, Nanjing Medical University, Nanjing, P. R. China.

ABSTRACT
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

No MeSH data available.


Related in: MedlinePlus

MiR-K3 enhances the activation of AKT in HUVEC by targeting GRK2.(A). Western blotting analysis of phosphorylated and total AKT in HUVEC transduced with mpCDH or miR-K3, and HUVEC treated with PBS (PBS) or infected with KSHV (KSHV), respectively. (B). Western blotting analysis of phosphorylated AKT in HUVEC transduced with mpCDH or miR-K3, which were further transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (C). Western blotting analysis for CXCR2 and phosphorylation levels of AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (D). Western blotting analysis of CXCR2 and the phosphorylation levels of AKT in KSHV-infected HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) and its control (mpCDH) or lentivirus-mediated miR-K3 sponge (miR-K3 sponge) and its control (pCDH), respectively. (E). Western blotting analysis of phosphorylation levels of AKT in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH). (F). Western blotting analysis of phosphorylation levels of AKT in HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and further with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2). (G). Western blotting analysis of phosphorylated AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH). (H). GRK2 physiologically hijacked AKT in GRK2-expressing HUVEC. HUVEC were transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE) and subjected to co-immunoprecipitation with the antibody against GRK2 (IP: Anti GRK2) or AKT (IP: Anti AKT) followed by Western blotting using indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581863&req=5

ppat.1005171.g007: MiR-K3 enhances the activation of AKT in HUVEC by targeting GRK2.(A). Western blotting analysis of phosphorylated and total AKT in HUVEC transduced with mpCDH or miR-K3, and HUVEC treated with PBS (PBS) or infected with KSHV (KSHV), respectively. (B). Western blotting analysis of phosphorylated AKT in HUVEC transduced with mpCDH or miR-K3, which were further transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (C). Western blotting analysis for CXCR2 and phosphorylation levels of AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (D). Western blotting analysis of CXCR2 and the phosphorylation levels of AKT in KSHV-infected HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) and its control (mpCDH) or lentivirus-mediated miR-K3 sponge (miR-K3 sponge) and its control (pCDH), respectively. (E). Western blotting analysis of phosphorylation levels of AKT in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH). (F). Western blotting analysis of phosphorylation levels of AKT in HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and further with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2). (G). Western blotting analysis of phosphorylated AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH). (H). GRK2 physiologically hijacked AKT in GRK2-expressing HUVEC. HUVEC were transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE) and subjected to co-immunoprecipitation with the antibody against GRK2 (IP: Anti GRK2) or AKT (IP: Anti AKT) followed by Western blotting using indicated antibodies.

Mentions: Since CXCR2 activated AKT signaling to promote the migration and invasion of lymphocytes and cancer cells [50,51], we asked whether AKT signaling was also involved in miR-K3 and KSHV induction of cell migration and invasion. Consistent with the previous reports [52], KSHV infection of HUVEC induced the phosphorylation of AKT (Fig 7A). Expression of miR-K3 also induced the phosphorylation of AKT in HUVEC (Fig 7A). Overexpression of GRK2 in miR-K3-expressing HUVEC dramatically inhibited AKT activation (Fig 7B). Similar results were also observed in KSHV-infected HUVEC, where ectopic expression of GRK2 led to the inhibition of AKT activation and a reduction of CXCR2 level (Fig 7C). In addition, overexpression of miR-K3 further enhanced AKT activation and increased the expression level of CXCR2 in KSHV-infected HUVEC while miR-K3 sponge effectively reduced the levels of phosphorylated AKT and CXCR2 expression (Fig 7D). As expected, knockdown of GRK2 with shRNAs was sufficient to increase the phosphorylated AKT level in normal HUVEC (Fig 7E). Because the expression level of CXCR2 was positively correlated with the level of AKT activation (Fig 7C and 7D), we examined the role of CXCR2 in AKT activation. Knock-down of CXCR2 decreased the level of phosphorylated AKT in either miR-K3-expressing or KSHV-infected HUVEC (Fig 7F and 7G). These observations implied that miR-K3/GRK2/CXCR2/AKT axis mediates miR-K3- and KSHV-induced cell migration and invasion.


A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling.

Hu M, Wang C, Li W, Lu W, Bai Z, Qin D, Yan Q, Zhu J, Krueger BJ, Renne R, Gao SJ, Lu C - PLoS Pathog. (2015)

MiR-K3 enhances the activation of AKT in HUVEC by targeting GRK2.(A). Western blotting analysis of phosphorylated and total AKT in HUVEC transduced with mpCDH or miR-K3, and HUVEC treated with PBS (PBS) or infected with KSHV (KSHV), respectively. (B). Western blotting analysis of phosphorylated AKT in HUVEC transduced with mpCDH or miR-K3, which were further transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (C). Western blotting analysis for CXCR2 and phosphorylation levels of AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (D). Western blotting analysis of CXCR2 and the phosphorylation levels of AKT in KSHV-infected HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) and its control (mpCDH) or lentivirus-mediated miR-K3 sponge (miR-K3 sponge) and its control (pCDH), respectively. (E). Western blotting analysis of phosphorylation levels of AKT in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH). (F). Western blotting analysis of phosphorylation levels of AKT in HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and further with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2). (G). Western blotting analysis of phosphorylated AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH). (H). GRK2 physiologically hijacked AKT in GRK2-expressing HUVEC. HUVEC were transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE) and subjected to co-immunoprecipitation with the antibody against GRK2 (IP: Anti GRK2) or AKT (IP: Anti AKT) followed by Western blotting using indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581863&req=5

ppat.1005171.g007: MiR-K3 enhances the activation of AKT in HUVEC by targeting GRK2.(A). Western blotting analysis of phosphorylated and total AKT in HUVEC transduced with mpCDH or miR-K3, and HUVEC treated with PBS (PBS) or infected with KSHV (KSHV), respectively. (B). Western blotting analysis of phosphorylated AKT in HUVEC transduced with mpCDH or miR-K3, which were further transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (C). Western blotting analysis for CXCR2 and phosphorylation levels of AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE). (D). Western blotting analysis of CXCR2 and the phosphorylation levels of AKT in KSHV-infected HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) and its control (mpCDH) or lentivirus-mediated miR-K3 sponge (miR-K3 sponge) and its control (pCDH), respectively. (E). Western blotting analysis of phosphorylation levels of AKT in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH). (F). Western blotting analysis of phosphorylation levels of AKT in HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and further with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2). (G). Western blotting analysis of phosphorylated AKT in normal HUVEC or KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH). (H). GRK2 physiologically hijacked AKT in GRK2-expressing HUVEC. HUVEC were transduced with lentivirus-GRK2 (GRK2) or its control (pHAGE) and subjected to co-immunoprecipitation with the antibody against GRK2 (IP: Anti GRK2) or AKT (IP: Anti AKT) followed by Western blotting using indicated antibodies.
Mentions: Since CXCR2 activated AKT signaling to promote the migration and invasion of lymphocytes and cancer cells [50,51], we asked whether AKT signaling was also involved in miR-K3 and KSHV induction of cell migration and invasion. Consistent with the previous reports [52], KSHV infection of HUVEC induced the phosphorylation of AKT (Fig 7A). Expression of miR-K3 also induced the phosphorylation of AKT in HUVEC (Fig 7A). Overexpression of GRK2 in miR-K3-expressing HUVEC dramatically inhibited AKT activation (Fig 7B). Similar results were also observed in KSHV-infected HUVEC, where ectopic expression of GRK2 led to the inhibition of AKT activation and a reduction of CXCR2 level (Fig 7C). In addition, overexpression of miR-K3 further enhanced AKT activation and increased the expression level of CXCR2 in KSHV-infected HUVEC while miR-K3 sponge effectively reduced the levels of phosphorylated AKT and CXCR2 expression (Fig 7D). As expected, knockdown of GRK2 with shRNAs was sufficient to increase the phosphorylated AKT level in normal HUVEC (Fig 7E). Because the expression level of CXCR2 was positively correlated with the level of AKT activation (Fig 7C and 7D), we examined the role of CXCR2 in AKT activation. Knock-down of CXCR2 decreased the level of phosphorylated AKT in either miR-K3-expressing or KSHV-infected HUVEC (Fig 7F and 7G). These observations implied that miR-K3/GRK2/CXCR2/AKT axis mediates miR-K3- and KSHV-induced cell migration and invasion.

Bottom Line: Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion.Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT.Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P. R. China; Key Laboratory Of Pathogen Biology Of Jiangsu Province, Nanjing Medical University, Nanjing, P. R. China; Department of Microbiology, Nanjing Medical University, Nanjing, P. R. China.

ABSTRACT
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

No MeSH data available.


Related in: MedlinePlus