Heller Brown Lab
Research
Joan Heller Brown, PhD
Professor & Chair
GPCRs in growth regulation
The brain contains a population of cells called astrocytes which serve to support neurons. Unlike neurons, astrocytes proliferate in response to brain injury and also express genes that mediate inflammation and cell growth. Aberrant astrocyte growth can lead to development of scar tissue, to inflammation, or to glioblastoma, a particularly aggressive form of cancer. We are examining novel mechanisms by which inflammatory mediators that act on G-protein coupled receptors (GPCRs) stimulate astrocyte proliferation and gene expression.
Thrombin, S1P and LPA are mediators of inflammation and potent activators of glial cell growth. We believe this occurs because these receptors couple to the oncogenic G-protein G12/13 which leads to activation of the small G-protein RhoA. We are studying two intriguing novel targets of RhoA. One is phospholipase C epsilon, unique in that it appears to localize to an intracellular compartment and to act as guanine nucleotide exchange factor for another small G-protein Rap1 which activates the oncogene BRaf.
A second signal transducer of considerable interest is Cyr61/CCN1 which is highly induced in glioblastoma, required for their proliferation, and acts as a secreted extracellular matrix protein that engages integrins, providing a cooperative mechanism for sustained mitogenic responses to GPCR stimulation.
We carry out molecular analysis on glioblastoma cell lines, using siRNA or lentiviral shRNA expression to knock down molecules of interest, as well as primary astrocytes isolated from brains of wild type and knockout mice. We assess changes in cell signaling events by Western blotting, by FRET based fluorescence imaging of second messengers and kinases, and by cell proliferation and apoptosis assays. For in vivo studies we examine the role of PLC epsilon and Cyr61 on growth of tumor cells and on tissue response to brain or spinal injury in mice.
Signaling pathways in heart diseases
When the heart is stressed by increased blood pressure of lack of oxygen it adapts by increasing the expression or activity of survival pathways. Our lab is interested in elucidating these pathways and the molecular mechanisms by which they work. We use isolated heart cells as a model system, and generate transgenic or knockout mice to probe the relevance of these signaling pathways to development of heart failure in vivo.
Of particular interest is why theses mechanisms fail, causing cardiomyocytes to die and making the heart unable to contract effectively and heart failure to develop. Is it loss of survival pathways, development of apoptotic/necrotic mediators, or a shift in the balance between these pathways? Candidate signaling molecules of particular interest to us are ligands for G-protein coupled receptors such as S1P, the heterotrimeric G-protein Gq and small G-protein RhoA, both of which turn on protective pathways that ultimately decompensate to pathways that induce apoptosis, the Ca regulated protein kinase CaMKII which regulates nuclear gene expression and Ca handling, and the protective molecule Akt, with targets at the mitochondria that control cell survival.
We address these questions in cultured cells using knockdown of molecules of interest with siRNA or overexpression by adenoviral vectors. We then translate these findings in vivo using knockout or transgenic mice which can be experimentally manipulated to induce heart failure. We measure changes in gene expression or protein expression/phosphorylation (Western blotting), visualize intracellular localization of molecules (confocal microscopy or FRET), detect cell death (staining or ELISA), measure intracellular Ca mobilization and mitochondrial integrity and evaluate heart function.