Evidence indicates that prolactin profoundly influences rat liver growth via receptor-mediated stimulation of PKC in the hepatocyte nucleus. Therefore, the hypothesis to be challenged by the experiments described in this proposal, is that prolactin, internalized by receptor-mediated endocytosis, is channelled to the nucleus, where it interacts with specific nuclear prolactin receptors coupled to PKC activation, which thereby evokes transcription of specific messenger RNAs requisite for a trophic response. This hypothesis will be tested in two distinctly different experimental cell systems, one transformed (Nb2 lymphoma cells) and one normal (rat hepatocytes) , in which prolactin stimulates growth. In both systems we will identify the nuclear prolactin receptor and investigate its coupling to mRNA transcription. By investigating two distinctly different paradigms of prolactin-induced mitogenesis, we will be able to determine whether the direct interaction of prolactin within the nucleus is phenomenon common to multiple systems. These studies will establish a direct nuclear site of action for growth factors such as prolactin and extend our understanding of the mechanism by which growth factors stimulate normal and tumor cell growth.
The specific aims are as follows: 1.) To determine whether prolactin, internalized by receptor-mediated endocytosis, is targeted to the hepatocyte and Nb2 lymphoma cell nucleus; 2.) To identify and characterize the prolactin receptor present within hepatocyte and Nb2 lymphoma cell nuclei; 3.) To determine whether nuclear prolactin receptor occupation leads to transcription of """"""""rapid response genes"""""""". We will study the intracellular fate of prolactin in intact cells Specific Aim 1) by indirect immunofluorescence and by subcellular fractionation of cells incubated with radiolabeled prolactin. The nuclear prolactin receptor will be identified and characterized (Specific Aim 2) by immunoprecipitation and ligand-cross-linking techniques and gel electrophoresis. Nuclear runoff and northern blot analysis will be utilized to determine which specific genes are transcribed in response to prolactin treatment in verifiably pure rat liver and Nb2 node lymphoma cell nuclei (Specific Aim 3). The elucidation of the mechanism(s) by which growth factors and hormones modulate cell proliferation will significantly broaden our understanding of normal and aberrant growth regulation. Since many transformed cells either synthesize and/or require growth factors for proliferation, the demonstration of a receptor-mediated nuclear site of action for these substances will suggest alternative cellular targets for possible therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29DK044439-03
Application #
2143784
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1992-09-30
Project End
1997-09-29
Budget Start
1994-09-30
Budget End
1995-09-29
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of North Dakota
Department
Pharmacology
Type
Schools of Medicine
DUNS #
102280781
City
Grand Forks
State
ND
Country
United States
Zip Code
58202
Krumenacker, J S; Buckley, D J; Leff, M A et al. (1998) Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression. Endocrine 9:163-70
Krumenacker, J S; Montgomery, D W; Buckley, D J et al. (1998) Prolactin receptor signaling: shared components with the T-cell antigen receptor in Nb2 lymphoma cells. Endocrine 9:313-20
Zhang, M; Buckley, D J; Lavoi, K P et al. (1998) Heat-induced proteolysis of HSF causes premature deactivation of the heat shock response in Nb2 lymphoma cells. Cell Stress Chaperones 3:57-66
Montgomery, D W; Krumenacker, J S; Buckley, A R (1998) Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation. Endocrinology 139:811-4
Gout, P W; Kang, Y J; Buckley, D J et al. (1997) Increased cystine uptake capability associated with malignant progression of Nb2 lymphoma cells. Leukemia 11:1329-37
Buckley, A R; Leff, M A; Buckley, D J et al. (1996) Alterations in pim-1 and c-myc expression associated with sodium butyrate-induced growth factor dependency in autonomous rat Nb2 lymphoma cells. Cell Growth Differ 7:1713-21
Leff, M A; Buckley, D J; Krumenacker, J S et al. (1996) Rapid modulation of the apoptosis regulatory genes, bcl-2 and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 137:5456-62
Rao, Y P; Buckley, D J; Olson, M D et al. (1995) Nuclear translocation of prolactin: collaboration of tyrosine kinase and protein kinase C activation in rat Nb2 node lymphoma cells. J Cell Physiol 163:266-76
Rao, Y P; Buckley, D J; Buckley, A R (1995) Rapid activation of mitogen-activated protein kinase and p21ras by prolactin and interleukin 2 in rat Nb2 node lymphoma cells. Cell Growth Differ 6:1235-44
Buckley, A R; Buckley, D J; Leff, M A et al. (1995) Rapid induction of pim-1 expression by prolactin and interleukin-2 in rat Nb2 lymphoma cells. Endocrinology 136:5252-9

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