My laboratory has focused on understanding growth and differentiation events in renal cells that are modulated by heterotrimeric guanine nucleotide binding (G) proteins.
Louis Ercolani, MD
Assistant Professor of Medicine
149 13th Street
Charlestown, MA 02129
Phone: (617) 726-5664
Fax: (617) 726-5669
We have determined that one G protein isoform, Gia-2 is critical to these processes and is encoded by a protooncogene. We have isolated and characterized this gene and a related isoform Gia-3 in renal cells and determined they are members of a very ancient supergene family with comparable homologues in more primitive eukaryotic cells. We have demonstrated there is a profound upregulation of the Gia-2 gene during growth and differentiation of LLC-PK1 renal cells that modulates a mitogenic cascade linked to downstream activation of p42/44 44 mitogen activated protein kinase. We have since expanded the scope of these studies to other cell types including pneumocytes. Gia-2 activation of this mitogenic cascase triggers the translocation of an early growth response gene product (EGR-1) to the nucleus allowing it to bind a cognate cis site in the 5' flank of the Gia-2 with resultant gene transactivation. Following differentiation, EGR-1 expression is extinguished and the site is occupied by a smaller molecular weight species, the Wilms' tumor suppressor which prevents further Gia-2 gene transactivation. Utilizing the LLC-PK1 cell as a unique renal model of WT1 function, we have since demonstrated that WT1 cis-binding as well as its transcription activities are critically dependent on an accessory protein first determined to play a role in prostatic apoptosis (Par-4).
In differentiated LLC-PKl cells, we have demonstrated the Gia-2 gene can be dynamically-regulated at other cis sites by different transcription factors. Among these are glucocorticoids which repress even basal activity of this gene. Alternatively, we have demonstrated cAMP can induce a "CAAT" box transcription factor which robustly transactivates the gene. This finding also provides solid evidence for an existing feedback loop mechanism to downregulate adenylyl cyclase activity in this cell type by increased production of Gia-2 peptide which is inhibitory for adenylyl cyclase. Utilizing yeast two-hybrid screening, we have recently identified novel and known effectors of Gia-2 action among these are tubulins and RGS family members which are known to modulate the activity of Gia-2 proteins as well as scaffold protein which can also function as a receptor for activated protein kinase C. We are currently examining which hormones influence these mitogenic cascades and have recently determined that Hepatocyte Growth Factor may play a key role in Gia-2 dependent differentiation processes in renal cells. In related studies utilizing the deduced crystal structure of the Gia-1, we have identified the residues in both Gqa and Gia-2 which are critical for the targeting of these molecules to differing membrane domains in polarized renal cells. We believe our ongoing structural and functional studies of the regulation of the Gia-2 gene and its product will continue to provide novel insights into renal development and physiology.
- Holtzman EJ, Kinane TB, West K, Soper BW, Karga IT, Ausiello DA, and Ercolani L. Transcriptional regulation of G-protein alpha i subunit genes in LLC-PK1 renal cells and characterization of the porcine G alpha 1-3 gene promoter. J. Biol. Chem. 1993. 268: 3964-3975.
- Kinane TB, Shang C, Finder JD, and Ercolani L. cAMP regulates G-protein alpha i-2 subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor. J. Biol. Chem. 1993. 268: 24669-24676.
- Kinane TB, Filider JD, Kawashima A, Brown D, Abbate M, Shang C, Fredericks WJ, Rauscher FJ, Sukhatme VP, and Ercolani L. Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein alpha i-2 protooncogene transcription. J. Biol. Chem. 1994. 269: 27503-27509.
- Kinane TB, Findler JD, Kawashima A, Brown D, Abbate M, Fredericks WJ, Sukhatme VP, Rauscher FJ, and Ercolani L. LLC-PK1 cell growth is repressed by WT1 inhibition of G-protein alpha i-2 protooncogene transcription. Biol. Chem. 1995. 270: 30760-30764.
- Kinane TB, Kang I, Chu A, Chin SH, and Ercolani L. Gai-2 mediates renal LLC-PK1 growth by a Raf independent activation of p42/p44 MAP kinase. Am. J. Physiol. 1997. 272: F273-F282.
- Kinane TB, Komatsuzaki K, Alexio MD, Sunday ME, and Ercolani L. Regulation of the G protein Galphai2 by growth and developmentin fetal airway epithelium. Am. J. Resp. Cell and Mol. Biol. 1999. 20(1): 35-42.