Research in my laboratory is directed towards elucidating the mechanisms of action of the vasoactive peptide angiotensin II (Ang II), and the growth factors insulin and platelet-derived growth factor(PDGF), and how they regulate proliferation and differentiated functions of cells and tissues. The major focus is on the control of protein kinase cascades, because phosphorylation is used repeatedly to regulate protein function. For example, previous work in my laboratory on cultured rat aortic vascular smooth muscle cells (VSMC) and phenotypically-similar glomerular mesangial cells, has shown that protein tyrosine phosphorylation plays a critical role in Ang II-mediated intracellular signaling cascades. This is true despite the fact that G protein-coupled receptors in general, and the Ang II, AT1 receptor in particular, possess no intrinsic tyrosine kinase activity. In addition, it is also now recognized that Ang II can act not only as a vasoactive peptide, but also as a growth factor. In particular, Ang II has been shown to stimulate proliferative and hypertrophic growth in VSMC, glomerular mesangial cells, cardiac fibroblasts, and myocytes via AT1 receptor binding. Like classic growth factors (e.g., platelet-derived growth factor [PDGF], insulin, epidermal growth factor [EGF]) and some cytokines (e.g., interferons and interleukins), Ang II is also capable of stimulating a rapid increase in the mRNA levels of c-fos, an early growth response gene implicated in VSMC proliferation. However, the Ang II-stimulated intracellular signaling cascades responsible for c-fos induction, and therefore, proliferation in VSMC and mesangial cells have not been well-defined. I believe that one candidate mitogenic signaling cascade involves the activation of the small GTP-binding protein, Ras, which is traditionally mediated via classic growth factor receptors. Ras activation promotes the formation of a membrane-bound complex with Raf-1 (a serine/threonine protein kinase). Subsequent tyrosine phosphorylation of Raf-1 leads to its activation and the sequential stimulation of several cytoplasmic protein kinases, collectively known as the mitogen-activated protein kinase (MAPK) pathway. This phosphorylation cascade in turn, activates a set of regulatory elements leading to the stimulation of early response genes and cellular growth. In support of this hypothesis, our laboratory has previously shown that as with classic growth factors, Ang II-induced protein tyrosine phosphorylation promotes the activation of p21ras in VSMC and rat kidney mesangial cells.

In addition to the MAP kinase pathway, my laboratory is also very actively studying another mitogenic cascade. This second mitogenic cascade which is activated by many cytokine receptors (e.g., interferons and interleukins) involves the JAK (Janus Kinases) family of cytoplasmic tyrosine kinases. JAK-mediated tyrosine phosphorylation of STAT (Signal Transducer and Activators of Transcription) family members promotes the translocation of these transcription factors to the nucleus, where they bind to specific DNA motifs and induce c-fos gene transcription. In VSMC, my laboratory has previously shown that Ang II stimulates the tyrosine phosphorylation of JAK isoforms (JAK2 and Tyk2), the tyrosine kinase activity of JAK2, and the tyrosine phosphorylation of STAT isoforms (STAT1, STAT2, and STAT3). Finally, we have recently shown that Ang II induces the formation of a complex between JAK2 and the AT1 receptor itself via the action of the tyrosine phosphatase SHP2, acting as an anchor protein.

In summary, my laboratory is actively studying the role of JAK/STAT and Ras/Raf-1/MAPK signaling cascades in the cellular proliferation mediated by activation of G protein-coupled AT1 receptor and classic growth factor receptors (e.g., PDGF and insulin). That is, we are focusing on several problems concerning both the JAK/STAT and MAP kinase pathways: the structure and functions of the MAP kinases, JAK kinases, STAT proteins, and the phosphatases that regulate their function; the diversity and specificity of their upstream activators; the physiological functions of these protein kinase cascades in VSMC, rat kidney mesangial cells and bovine aorta endothelial cells; and the mechanisms by which these kinase networks cascades are regulated.

Grant Support as Principal Investigator

1993 - 1995 NIH Minority Supplemental Award, Emory University, $150,000 Direct costs.

1994 - 1999 American Heart Association, National Center, Minority Developmental Award, $300,000 Direct costs.

1994 - 1996 American Heart Association, Georgia Affiliate, Grant-In-Aid Award, $66,000 Direct costs.

1997 - 2002 NIH R01 First Award, $350,000 Direct costs.

1997 - 2002 NIH P01 Project Grant, Dr. Douglas C. Eaton Director, $4,000,000 Direct costs.

1997 - 1999 American Heart Association, National Center, Grant In Aid, $150,000 Direct costs.

2000 - 2003 American Heart Association, National Center, Established Investigator Award, $300,000 Direct costs.

Honors and Awards

Invited Speaker

1995 Epithelium Cell Signaling Symposium, FASEB, Atlanta, Georgia

1995 Gordon Conference on Angiotensin II, El Ciocco, Italy

1995 Dept. of Physiology, Emory University, Atlanta, Georgia

1996 Cellular Signaling Symposium, Biophysical Society, Baltimore, MD

1996 American Society of Nephrology, New Orleans, LA

1997 Biomedicine ‘97 Meeting, Washington, DC

1999 Moorehouse School of Medicne, Atlanta, GA

2000 International Society of Hypertension, Chicago, Ill

2001 Dept. of Physiology and Biophysics, Columbia University, New York, NY

Publications

Marrero, M.B., Paxton, W.G., Duff, J.L., Berk, B.C., Bernstein, K.E. (1994) Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-(1 in vascular smooth muscle cells. J. Biol. Chem., 269, 10935-10939.

Marrero, M.B., Schieffer, B., Paxton, W., Schieffer, E., and Bernstein, K.E. (1995) Electroporation of pp60c-src antibodies inhibits the angiotensin II activation of phospholipase C-(1 in vascular smooth muscle cells. J. Biol. Chem., 270, 11230-11235.

Marrero, M.B., Schieffer, B., Paxton, W.G, Heerdt, L., Berk, B.C., Delafontaine, P., and Bernstein, K.E. (1995) Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature, 375, 247-250.

Schieffer, B., Paxton, W., Chai, Q., Marrero, M.B. and Bernstein, K.E. (1996) Angiotensin II regulates the activity of p21ras via pp60c-src in vascular smooth muscle cells. J. Biol. Chem., 271, 10329-10333.

Marrero, M.B., Schieffer, B., Bernstein, K.E., Ma, H., and Ling, B.L. (1996) Angiotensin II-induced tyrosine phosphorylation stimulates phospholipase C-(1 and CL- channels in glomerular mesangial cells. Am. J. Physiol., 270, C1834-C1842.

Harp, J.B., Sayeski, P.P., Scanlon, M., Bernstein, K.E., and Marrero, M.B. (1997) Role of intracellular calcium in the angiotensin II-mediated tyrosine phosphorylation and dephosphorylation of PLC-(1. Biochem. Biophys. Res. Commun., 232, 540-544.

Ali, M.S., Schieffer, B., Delafontaine, P., Bernstein, K.E., Ling, B.N., and Marrero, M.B. (1997) Angiotensin II stimulates the tyrosine phosphorylation and activation of IRS-1 and PTP-1D in vascular smooth muscle cells. J. Biol. Chem., 272, 12373-12379.

Schieffer, B., Drexler, H. Ling, B.N., and Marrero, M.B. (1997) G protein-coupled receptors control vascular smooth cell proliferation via pp60c-src and p21ras. Am. J. Physiol., 41, C2019-C2030.

Ali, M.S., Hayzer, D., Dirsken, L.B., Sayeski, P.P., Marrero, M.B., and Bernstein, K.E. (1997) Dependence of the motif YIPP for the physical association of JAK2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor. J. Biol. Chem., 272, 23382-23388.

Marrero, M.B., Schieffer, B., Li, B., Sun, J., Harp J.B., and Ling B.N. (1997) Role of JAK/STAT and MAP kinase cascades in angiotensin II- and PDGF-induced vascular smooth muscle cell proliferation. J. Biol. Chem., 272, 24684-24690.

Bernstein, K.E., Ali, M.S., Sayeski, P.P., Semeniuk, D., and Marrero, M.B. (1998) New insights into the cellular signaling of seven transmembrane receptors: the role of tyrosine phosphorylation. Lab. Invest., 78, 3-7.

Venema, R.C., Ju, H., Venema, V.J., Schieffer, B., Harp, J.B., Ling, B.N., Eaton, D.C., and Marrero, M.B. (1998) Angiotensin II-induced association of PhospholipaseC-(1 with the G-protein-coupled AT1 receptor. J. Biol. Chem., 273, 7703-7708.

Marrero, M.B., Venema, R.C., Ma, H., Ling, B.N., and Eaton, D.C. (1998) Erythropoietin receptor-operated Ca2+ channels: activation by phospholipase C-(1. Kidney Int. 53, 1259-1268.

Lea, J.P., Ertoy, D., Hollis, J.L., Marrero, M.B., and Sands, J.M. (1998) Immunolocalization of phospholipase C isoforms in rat kidney. Kidney Int. 54, 1484-1490.

Marrero, M.B., Venema, V.J., Ju, H., Eaton, D.C., and Venema, R.C. (1998) Regulation of Angiotensin II-induced JAK2 tyrosine phosphorylation. Roles of SHP1 and SHP2. Am. J. Physiol. 275, C1216-C1223.

Venema, R.C., Venema, V.J., Eaton, D.C., and Marrero, M.B. (1998) Angiotensin II-induced tyrosine phosphorylation of signal transducers and activators of transcription 1 is regulated by janus-activated kinase 2 and Fyn kinases and mitogen-activated protein kinase phosphatase 1. J. Biol. Chem. 273, 30795-30801.

Marrero, M.B., Venema, V.J., He, H., Caldwell, R.B., and Venema, R.C. (1998) Inhibition by the JAK/STAT pathway of IFN(- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 252, 508-512.

Liang, H., Venema, V.J., Wang, X., Ju, H., Venema, R.C., and Marrero, M.B. (1999) Regulation of angiotensin II-induced phosphorylation of STAT3 in vascular smooth muscle cells. J. Biol. Chem. 274, 30795-30801.

He, H., Venema, V.J., Gu, X., Venema, R.C., Marrero, M.B., and Caldwell, R.B. (1999) Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through Flk-1/KDR activation of c-Src. J. Biol. Chem. 274, 25130-25135.

Marrero, M.B., Venema, V.J., Ju, H., He, H., Liang, H., Caldwell, R.B., and Venema, R.C. (1999) Endothelial nitric oxide synthase interactions with G-protein-coupled receptors. Biochem. J. 343, 335-340.

Amiri, F., Venema, V.J., Wang, X., Ju, H., Venema, R.C., and Marrero, M.B. (1999) Hyperglycemia enhances angiotensin II-induced JAK/STAT signaling in vascular smooth muscle cells. J. Biol. Chem. 274, 32382-32386.

Deng, J., Hua, K., Walter, A.W., Lesser, S.S., Greiner, A.H., Marrero, M.B., and Harp, J.B. (2000) Differential activation of signal transducer and activator of trancription 6 variants by fibroblast growth factor-2 and interleukin-4 in 3T3-L1 preadipocytes. Biochem. Biophys. Res. Commun. 267, 516-520.

Bartoli, M., Gu, X., Tsai, N.T., Venema, R.C., Brooks, S.E., Marrero, M.B., and Caldwell, R.B. (2000) VEGF activates signal transducers and activators of transcription proteins in aortic endothelial cells. J. Biol. Chem. 275, 33189-33192.

Marrero, M.B., Shaw. S., Wang, X., Amiri, F., and Eaton, D.C. (2001) Inhibition of JAK2 prevents the glucose-induced increased in TGF-beta and fibronectin synthesis in mesangial cells. Am. J. Physiol. In Press.

Amiri, F., Shaw, S., Wang, X., Eaton, D.C., and Marrero, M.B. (2001) Angiotensin II activation of the JAK/STAT pathway in rat kidney mesangial cells is altered by hyperglycemia. Kidney Int. In Press.

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