Involvement of c-src in β-Casein Expression by Mammary Epithelial Cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 241, 710–713 (1997)ARTICLE NO. RC977879

Involvement of c-src in b-Casein Expression byMammary Epithelial Cells

Peter Sørensen and Lewis G. Sheffield1

Dairy Science Department and Endocrinology-Reproductive Physiology Program,University of Wisconsin, Madison, Wisconsin 53706

Received November 13, 1997

transcriptional activation. JAK2 mediated STAT acti-HC11 mouse mammary epithelial cells were stably vation has been found to be essential for prolactin ac-

transfected with c-src or a dominant negative mu- tion but may not be sufficient for prolactin action (12).tant of c-src driven by a MMLV promoter. Prolactin In addition to JAK2, prolactin has been proposed toincreased c-src activity in control and c-src trans- activate a variety of other signaling pathways. In Nb2fected cells, while dominant negative c-src reduced cells, the prolactin receptor has been found to associatethe prolactin-induced increase in c-src activity.

with members of the src family of protein kinases, par-Dominant negative c-src also reduced the ability ofticularly fyn (13). In liver tissue, the prolactin receptorinsulin, hydrocortisone, and prolactin to induce b-has been found to associate with c-src (14). Memberscasein accumulation to levels one-half to one-thirdof the src family of protein kinases have been knownthat of control cells. This effect was uncorrelatedto affect a wide variety of cellular processes, includingwith any change in cell proliferation or laminin ac-growth (15), motility (16), morphogenesis (17) and cy-cumulation and was observed both in cultures growntoskeletal organization (18). However, whether the srcto confluency in the presence of EGF and in culturesfamily of kinases plays a functional role in mediatinggrown on laminin. q 1997 Academic Press

prolactin-induced milk protein gene expression is un-clear. Therefore, the objective of this study was to de-termine if inhibiting c-src activity in mammary epithe-

A combination of insulin, glucocorticoids and prolac- lial cells altered hormone-induced milk protein accu-tin are well established as minimal hormonal require- mulation.ments for the induction of milk protein gene expressionin mammary tissue in vitro (1). However, the molecular

MATERIALS AND METHODSmechanisms by which these hormones act remains un-clear. The development of established cells lines which Cell culture. HC11 cells were obtained from Dr. J. Rosen (Baylorrespond to appropriate lactogenic hormones and are College of Medicine, Houston, TX), with permission of the line’s origi-

nator, Dr. B. Groner (Institute for Experimental Cancer, Freiburg,readily manipulated in culture have accelerated prog-Germany) (2). Cells were routinely cultured in RPMI-1640 containingress in this area. One such line is HC11, a mouse mam-10 ng/ml EGF, 1 mg/ml insulin and 10% fetal bovine serum.mary epithelial cell line that can be induced to produce

Transfection. C-src and dominant negative c-src constructs wereb-casein when grown to confluency in the presence ofobtained from Dr. S.L. Warren (Institute for Cancer Research, Phila-EGF (2) or when grown on a laminin substratum (3).delphia, PA, 16). Plasmids were grown in E. coli DH5a and cells

Although the prolactin receptor is not a tyrosine ki- transfected by electroporation. Approximately 107 cells were sus-nase (4), prolactin has been shown to increase tyrosine pended in 0.5 ml HBSS along with 10 mg of plasmid DNA in a 0.5

cm electroporation cuvet. They were electroporated with a BTX 600phosphorylation (5, 6). Tyrosine phosphorylation iselectroporation apparatus using a field strength of 2.5 kV/cm, set onthought to be essential for prolactin-induced lactogen-ice for 10 minutes and returned to growth media. Cells were thenesis (7). Janis kinase 2 (JAK2) has been identified as selected with 400 mg/ml G418 sulfate for a period of 4 weeks. G418

a major prolactin receptor associated tyrosine kinase sulfate was removed from cultures when plating for experiments.(8, 9). This kinase is thought to phosphorylate members

src activity estimation. Cells were plated and grown as describedof the STAT family of transcription factors (particu- above, then treated with prolactin (1 mg/ml) for 15 minutes. Cellslarly STAT 5 in mammary tissue, 10, 11) and lead to were lysed with lysis buffer (10 mM Tris, pH 7.6, 5 mM EDTA, 50

mM NaCl, 30 mM Sodium pyrophosphate, 30 mM NaF, 0.1% BSA,0.5% Sodium Deoxycholeate 1% Triton X-100, 0.1% SDS and 1 mMPMSF), centrifuged at 14,000 g for 10 minutes at 47C and c-src immu-1 To whom correspondence should be addressed at 1675 Observa-

tory Drive, University of Wisconsin, Madison, WI 53706. noprecipitated from supernatant by incubating for 2 hours with anti-

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 241, No. 3, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c-src (UBI, Lake Placid, NY) and agarose conjugated protein A andG (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitateswere rinsed 4 times with lysis buffer lacking BSA, then suspendedin 10 ml dilution buffer (200 mM HEPES, pH 7.0, 10% glycerol, 0.1%NP40). To this was added 5 ml kinase assay buffer (250 mM Tris, pH7.0, 125 mM MgCl2, 25 mM MnCl2 and 0.25 mM Na3VO4). Enolase (5mg) was added as a substrate, and 20 mCi g32P-ATP (100 Ci/mmole,DuPont, Boston, MA) added. Reactions were continued for 20 min-utes at room temperature and stopped by adding 50 ml SDS loadingbuffer. After heating in a steam bath for 5 minutes, samples wereresolved by SDS-PAGE (19), dried, exposed to X-ray film and bandintensity determined by computer assisted densitometry (Collage,Fotodyne, New Berlin, WI).

Induction of lactation. Cells were plated onto 35 mm dishes (106

cells per dish) and grown until confluent. The day after confluency,the media was changed from growth media to lactogenic media, con-sisting of RPMI 1640 supplemented with 10% gelding serum, 1 mg/ml insulin, 1 mM hydrocortisone and 1 mg/ml prolactin. After 24hours, cells were lysed with SDS loading buffer and proteins sepa-rated by SDS-PAGE using a 12% separating gel (19), then trans-ferred to PVDF membranes (20). Membranes were blocked with 50mM phosphate, pH 7.0 containing 150 mM NaCl, 3% Tween-20 (PBS-T) and 2% BSA. Membranes were then probed with rabbit anti- FIG. 1. Induction of c-src kinase activity in parental HC11 cellsmouse mixed caseins (kindly provided by Dr. M.C. Neville, Univer- (CH11) and cell expression wild type c-src (SRC) or dominant nega-sity of Colorado Health Sciences Center, Bolder, CO), rinsed with tive c-src (DN). Cells were treated as controls or with 1 mg/ml prolac-PBS-T, secondary antibody conjugated with peroxidase (Sigma tin and src activity was measured after 15 minutes as described inChemical Co., St. Louis, MO) added, rinsed with PBS-T and devel- Materials and Methods. Mean { SEM of 4 determinations.oped using Chemiluminescence (DuPont). Band intensity was esti-mated using computer assisted image analysis (Collage). As an alter-native to the above, cells were also plated onto 35 mm dishes coatedwith laminin (UBI, 10 mg/ml) and treated as described above. DISCUSSION

Western blot analysis. Cells were plated as described above. Post-Results of these studies suggest that prolactin in-confluent cells were lysed with SDS loading buffer and proteins sepa-

rated by SDS-PAGE. Western blots were prepared as above and duces activation of c-src in mammary epithelial cells.probed with anti-c-src (UBI) and anti-laminins (Collaborative Bio- Previous results (14) have demonstrated association ofmedical Products, Bedford, MA) as described above. c-src with the prolactin receptor in liver tissue, and its

activation by prolactin. Similarly, the src-like kinaseRESULTS fyn has been found to be associated with the prolactin

receptor and activated by prolactin in NB2 ratlymphoma cells (13). Although the src family of kinasesWild-type HC11 cells exhibited a 5 fold increase in

c-src activity following treatment with lactogenic hor- have been implicated in a wide variety of physiologicalfunctions, including cell growth, motility, morphogene-mones (Fig. 1). Expression of wild-type c-src increased

basal levels of c-src activity, but the ability of prolactin sis and cytoskeletal organization (15-18), a role in pro-lactin action has not been clearly identified.to activate c-src was unaffected. In cells transfected

with a dominant negative c-src mutant, baseline c-src By using a dominant negative mutant of c-src, wedemonstrated that reduced c-src activation by prolactinactivity was decreased such that less than a 2 fold in-

crease was observed in response to prolactin (Fig. 1). leads to reduced b-casein accumulation in response tolactogenic hormones. Interestingly, mice expressing aIn addition, cell morphology, population doubling time

of HC11 cells and confluent cell density were not af- null mutant of c-src (21) have not been reported toexhibit abnormalities in lactation. However, the srcfected by transfection with wild-type or dominant nega-

tive c-src (not shown). family of enzymes appears to be redundant, and lackof an effect of ablation of one member may not indicateWhile cell growth was unaffected by c-src inhibition,

b-casein accumulation in response to lactogenic hor- lack of role for that member (22).Another interesting observation is that overex-mones was decreased in cells expressing dominant neg-

ative c-src, but not in cells expressing wild-type c-src pression of the wild-type c-src did not alter hormoneinduced b-casein production. A previous study (23) sug-(Fig. 2). However, b-casein expression was still detect-

able, albeit at levels approximately half that expressed gested that overexpression of src decreased differentia-tion of mammary epithelial cells. However, that studyin parental cells. Similar results were obtained if cells

were grown past confluency on plastic or if they were used a more active v-src, rather than the c-src constructused in these studies. Other studies have also reportedplated on a laminin matrix. In addition, dominant neg-

ative src expression did not alter laminin accumulation different responses to v-src than c-src (24).The mechanism by which c-src might act to in-in cell cultures (not shown).

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FIG. 2. b-Casein content of parental HC11 mammary epithelial cells or cells transfected with wild type or dominant negative c-src.Cells were grown and cultured in lactogenic media as described in Materials and Methods. Mean { SEM of data from 4 experiments.

authors thank Tyler Sisk, Susan Pederson, and Josie Lewandowskicrease milk protein gene expression remains unclear.for technical assistance, Dr. Steven Warren for the src plasmids, Dr.The major trans acting factor regulating b-casein ex-Berend Groner for the HC11 cells, and Dr. Margaret Nevelle for thepression is thought to be STAT 5, which is phosphory- anti-mouse caseins.

lated and activated by JAK2 in response to prolactin(10, 11). Glucocorticoid receptor and the transcrip- REFERENCEStion inhibitor YY1 are also important (25, 26). None

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Involvement of c-src in β-Casein Expression by Mammary Epithelial Cells