GENES RESPONSIVE TO GROWTH FACTORS 
ViKAS R SUKHATME, M.D., PH.D., Assistant InvesUgutor 
The primary focus of Dr. Sukhatme's laboratory is 
on characterizing genes that are growth factor in- 
ducible and encode transcription factors. The ex- 
pectation is that genes of this type will play broad 
roles as "nuclear transducers" by coupling early 
biochemical events that follow within seconds of 
ligand-receptor binding to long-term cellular re- 
sponses required for proliferation and differentia- 
tion. Until recently, c-fos was the only example of 
such a gene. Now work from several laboratories 
indicates that there are at least three other sets of 
early response genes that are likely to encode tran- 
scription factors: Egr-1, -2, and -3; c-jun and junB; 
and nurll. The Egr family discovered in Dr. 
Sukhatme's laboratory are zinc finger proteins of 
the Cys^-His^ class. The long-term goal of these 
studies is to understand the role of these gene 
products in cellular proliferation, as well as in sig- 
nal transduction and cellular differentiation. 
\. Egr-1. 
A. Is Egr-1 induced in every mitogenic process? Ear- 
lier work from Dr. Sukhatme's laboratory identified a 
cDNA designated Egr-1, also referred to as NGFI-A, 
zif268, and Krox24, respectively, by the groups of 
Drs. J. Milbrandt (Washington University), Daniel Na- 
thans (HHMI, The Johns Hopkins University), and R. 
Bravo (European Molecular Biology Laboratory, Hei- 
delberg), who have discovered the same gene inde- 
pendently. This transcript was growth factor induc- 
ible in diverse cell types by various mitogens. To 
date, Egr-1 induction has been noted upon mito- 
genic stimulation of murine and human fibroblasts, 
rat liver H35 hepatoma cells, rat renal mesangial and 
glomerular cells, monkey kidney BSC- 1 cells, murine 
splenic B cells and T cell clones, and human periph- 
eral blood lymphocytes, as well as in liver regenera- 
tion. Different mitogens, including many cell-type- 
specific ones (e.g., anti-(x antibody for B cells), were 
used. These data show the ubiquitous nature of 
Egr-1 induction during the onset of growth. Dr. 
Sukhatme's laboratory is currently asking whether 
there is a causal relationship; i.e., isEgr-1 stimulation 
sufficient and/or necessary for mitogenesis? Is Egr-1 a 
proto-oncogene? 
B. What changes in a cell's environment lead to 
Egr-1 induction? What intracellular pathways are 
used? Why is Egr-1 often co-regulated with c-fos? 
The emphasis here is on understanding the 
events — from cell surface to nucleus — that stimu- 
late Egr-1. Diverse mitogens induce Egr-1 mRNA. 
Because these mitogens act through different sec- 
ond messenger systems, it appears that Egr-1 stimu- 
lation lies at the convergence of these various path- 
ways. Furthermore, since these second messengers 
are generated by processes unrelated to cell 
growth, Egr-1 modulation may be more generally 
coupled to changes in a cell's extracellular environ- 
ment, e.g., by ionic changes, hormones, or neuro- 
transmitters. Dr. Sukhatme's previous work had 
shown that Egr-1 is a nerve growth factor-inducible 
gene in rat PC 12 cells, and cellular depolarization 
in vitro and seizure activity in vivo can induce its 
expression. During the last year these observations 
have been extended to other ligand-receptor inter- 
actions. In collaboration with Dr. K. Chien (Univer- 
sity of California at San Diego), it has been found 
that both a- and ^-adrenergic stimulation of cardiac 
myocytes causes striking Egr-1 expression, as does 
renal ischemia [collaboration with Dr. J. Bonventre 
(Harvard Medical School)]. Observations such as 
these are being extended to other systems so as to 
define the broad range of proximal events— from 
extracellular ligand-receptor interactions to intra- 
cellular second messengers— that modulate Egr-1 
expression. 
With regard to the latter, initial studies in Dr. 
Sukhatme's laboratory have been in fibroblasts. Ad- 
dition of the phorbol ester 12-O-tetradecanoylphor- 
bol-13-acetate (TPA) to these cells induces Egr-1 
mRNA. However, even in cells in which protein ki- 
nase C (PKC) has been downregulated, Egr-1 can be 
induced by epidermal growth factor (EGF) and by 
serum. Thus multiple intracellular pathways in fi- 
broblasts converge to induce Egr-1. To define the 
cis-acting elements that mediate these inductions, 
Dr. Sukhatme's laboratory recently cloned and se- 
quenced the Egr-1 gene, identified the transcription 
initiation site, and sequenced ~1 kb of upstream 
sequence. Of note are several putative cAMP re- 
sponse elements and API-binding sites as well as 
six so-called CArG boxes that form the inner core of 
the fos serum response element (SRE). Chloram- 
phenicol acetyltransferase (CAT) assays in fibro- 
blasts utilizing deletion mutants of the 5' regula- 
tory region suggest that these elements are 
responsible both for the TPA induction and serum 
inducibility of Egr-1. Gel shift assays show that 
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