Characterization of Stat5a and Stat5b homodimers and heterodimers and their association with the glucocortiocoid receptor in mammary cells

Abstract

The lactogenic hormones, i.e., prolactin and glucocorticoids, act in concert to stimulate transcription factors responsible for hormone-dependent milk protein gene expression. In the mammary gland, prolactin activates Stat5a and Stat5b and glucocorticoids activate the glucocorticoid receptor (GR). Immunoprecipitation experiments revealed that in mammary cells, Stat5a, Stat5b, and the GR are physically associated in vivo. The association is not dependent on lactogenic hormone treatment and is evident at all stages of mammary gland development. Immunodepletion experiments indicated that a fraction of GR and Stat5 proteins are not associated, suggesting that there are different intracellular pools of these proteins. Lactogenic hormone treatment of HC11 mammary cells resulted in tyrosine phosphorylation of Stat5a and Stat5b, dimerization, and rapid nuclear translocation of both Stat5 proteins. Following hormone treatment, Stat5a-Stat5b heterodimers were detected by their coimmunoprecipitation. In addition, immunodepletion experiments followed by gel shift analyses revealed the presence of active Stat5a and Stat5b homodimers. In mammary cells, Stat5b homodimers are less abundant than Stat5a homodimers. Although the GR does not bind the Stat5 DNA binding site directly, it could be detected with the Stat5-DNA complex. These results suggest that glucocorticoids affect milk protein gene expression via association of the GR with Stat5. Thus, there is a functional coupling between Stat-dependent and nuclear hormone receptor-dependent gene transcription.

FIG. 1

Stat5a and Stat5b DNA binding in mammary cells (EMSA). (A) A 6-μg amount of HC11 nuclear protein extract prepared from cell cultures treated for the indicated times with lactogenic hormones (lanes 2 to 7) or noninduced cells (lane 1) was incubated with radiolabeled oligonucleotide containing the Stat5 binding site. Complexes were electrophoresed in a 4% polyacrylamide native gel. (B and C) EMSA performed in the presence of the indicated antisera by using 6 μg of nuclear protein extracts from HC11 cells treated with lactogenic hormones for 7 min or 1 μg of nuclear protein extracts from lactating mammary gland, respectively. Prolactin, insulin, and dexamethasone were at concentrations of 5 μg/ml, 5 μg/ml, and 1 μM, respectively.

FIG. 2

Stat5a-Stat5b activation, nuclear translocation, and dimerization in HC11 cells. HC11 cells were treated with lactogenic hormones for the indicated times. A 500-μg amount of the nuclear and cytoplasmic protein fractions was immunoprecipitated (ip) with anti-Stat5a (A)- or anti-Stat5b (B)-specific antiserum. Immunocomplexes were resolved by SDS-8% PAGE. Filters were sequentially probed for phosphotyrosine (P-Y), Stat5a, and Stat5b by using specific antisera. Following each incubation, the membrane was stripped as described in Materials and Methods. b*, tyrosine-phosphorylated form of Stat5b. Molecular mass markers (in kilodaltons) are indicated on the right side. In the Western analysis for Stat5b, the upper bands in panels A (top panel) and B (lowest panel) are due to a nonspecific protein. Prolactin, insulin, and dexamethasone were at concentrations of 5 μg/ml, 5 μg/ml, and 1 μM, respectively. WB, Western blot; d, days.

FIG. 3

Active Stat5a and Stat5b homodimers in HC11 cells. A 200-μg amount of nuclear protein extracts from HC11 cells treated for 7 min with lactogenic hormones was immunodepleted (immunodepl.) of Stat5a or Stat5b as indicated; 6 μg of immunodepleted extracts was analyzed by EMSA either in the absence (control [C]) or in the presence of Stat5a or Stat5b antiserum as indicated. Complexes were resolved in a 4% polyacrylamide native gel. Complexes containing Stat5a or Stat5b and their supershifts are indicated. Prolactin, insulin, and dexamethasone were at concentrations of 5 μg/ml, 5 μg/ml, and 1 μM, respectively.

FIG. 4

Active Stat5a and Stat5b homodimers in lactating mammary glands. Nuclear protein extracts from mammary glands lactating for 10 days were immunodepleted of Stat5a or Stat5b as indicated. A 6-μg amount of immunodepleted (immunodepl.) extracts was analyzed by EMSA either in the absence (control [C]) or in the presence of Stat5a or Stat5b antiserum as indicated. Stat5a- or Stat5b-DNA complexes are indicated by arrows on the right side. Immunodepleted extracts gave a background band which masked the Stat5a and Stat5b supershifts. The electrophoretic mobility of the Stat-DNA complex from nonimmunodepleted nuclear extract is shown in lane 1.

FIG. 5

Stat5-GR association in mammary cells. (A) HC11 cells were treated for the indicated times with different combinations of lactogenic hormones (dexamethasone [D], insulin [I], and prolactin [P]). Control cells (−) were left in serum-free medium. A 500-μg amount of whole-cell protein extracts was immunoprecipitated (immunoppt.) with GR antiserum and subjected to SDS-10% PAGE. The membrane was successively incubated with Stat5b and GR antisera. (B) A 500-μg amount of whole-cell protein extracts of mammary gland tissues prepared from glands of virgin, 18-day-pregnant, 1-day-lactating, 10-day-lactating, and 15-day-lactating mice and from 1-day-involuting glands was immunoprecipitated with GR antiserum and analyzed as described in above. (C) A 500-μg amount of whole-cell protein extracts of mammary gland tissues prepared from glands of virgin, 18-day-pregnant, 1-day-lactating, and 10-day-lactating mice and from 1-day-involuting glands were immunoprecipitated with Stat5a antiserum. The membrane was successively incubated with GR and Stat5a antisera. (D) A 80-μg amount of GR-immunodepleted extracts was resolved by SDS-8% PAGE and incubated with Stat5a and Stat5b antisera. Prolactin, insulin, and dexamethasone were at concentrations of 5 μg/ml, 5 μg/ml, and 1 μM, respectively. WB, Western blot; d, days; preg, pregnant; lact, lactating; invol, involuted.

FIG. 6

Protein levels of Stat5a, Stat5b, and GR in in vitro-cultured cells and organs. (A) An 80-μg amount of whole-cell extracts made from HC11 cells, lactating mammary gland (MG), liver, and NIH 3T3 cells was resolved by SDS-8% PAGE. The membrane was probed for Stat5a (left side) or Stat5b (right side) (upper panel), stripped, and reprobed for GR (lower panel) with specific antisera. (B) The membranes were stained with 0.1% amido black to control for protein loading.

FIG. 7

Stat5-GR association in in vitro-cultured cells and organs. A 500-μg amount of whole-cell extracts made from HC11 cells, lactating mammary gland (MG), liver, and NIH 3T3 cells was immunoprecipitated (ip) with Stat5a antiserum (A) or Stat5b antiserum (B) and resolved by SDS-10% PAGE. The membrane was incubated sequentially with GR and Stat5a (A) or Stat5b (B) antiserum. (C) An 80-μg amount of the protein extracts described above (lanes 1 to 4) or 500 μg of the same extracts immunoprecipitated with GR antiserum (lanes 5 to 8) was resolved by SDS-10% PAGE and probed for Stat3 with specific antiserum. Arrows indicate the proteins shown on the Western Western blot (WB). Ig, immunoglobulin.

FIG. 8

Stat5 and GR nuclear translocation. (A) HC11 cells were treated for the indicated times with different combinations of lactogenic hormones (dexamethasone [D], insulin (I), and prolactin [P]). Control cells (−) were left in serum-free medium. Nuclear and cytoplasmic fractions were prepared, and 80 μg of each was subjected to SDS-8% PAGE and probed with Stat5a and GR antisera. (B) HC11 cells were treated for the indicated times with lactogenic hormones; 80 μg of whole-cell protein extracts was subjected to SDS-8% PAGE and incubated with Stat5a and Stat5b antisera. (C) Cellular fractionation was controlled by probing two independent preparations of each fraction with a Raf-1-specific antiserum. Prolactin, insulin, and dexamethasone were at concentrations of respectively, 5 μg/ml, 5 μg/ml, and 1 μM, respectively.

FIG. 9

GR antiserum immunoprecipitates the Stat5-DNA complex. HC11 cells were treated for 7 min with lactogenic hormones, and 6 μg of nuclear protein extract was incubated in EMSA buffer with radiolabeled mutated (MT) or wild-type (WT) Stat5 binding site. Anti-GR or preimmune serum was added to the reaction and immunoprecipitated with protein A-Sepharose beads. Immunocomplexes were collected, and radioactivity was quantified in a scintillation counter. Values were expressed as percentages of initially added probe. Prolactin, insulin, and dexamethasone were at concentrations of 5 μg/ml, 5 μg/ml, and 1 μM, respectively.