Involvement of SOCS-1, the Suppressor of Cytokine Signaling, in the ...

Involvement of SOCS-1, the Suppressor of Cytokine Signaling, in the Prevention of ProlactinResponsive Gene Expression in Decidual Cells

Uriel Barkai, Anne Prigent-Tessier, Christian Tessier, Gil B. Gibori, and Geula Gibori Department of Physiology and Biophysics College of Medicine University of Illinois Chicago, Illinois 60612

tiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the ␣2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of ␣2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells. (Molecular Endocrinology 14: 554–563, 2000)

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent ␣2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of ␣2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal ␣2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce ␣2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the ␣2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, ␣2-macroglobulin mRNA expression was ini-

INTRODUCTION In the rat, decidualization of uterine stromal cells, induced by either implantation or artificial stimuli, produces two regions of transformed cells, the mesometrial and antimesometrial decidua, which differ in cellular morphology, physiological characterization, and protein production (1–4). The major protein produced by the mesometrial cells is ␣2-macroglobulin (␣2-MG) (5, 6), a protease inhibitor, which appears to play an important role in limiting trophoblast invasion (7–11) and whose expression is induced in several target tissues, including the decidua, by PRL and PRLrelated hormones (6, 12–14). Yet despite the fact that the antimesometrial cells produce PRL and PRLrelated hormones and that the PRL-receptor (PRL-R) is present in both cell types (15), only the mesometrial cells express the ␣2-MG gene. The reason why ␣2-MG expression is silenced in the antimesometrial cells is not clear. One key event governing the transduction of the PRL signaling is well characterized: the presence of the effector induces membrane receptor dimerization which leads to transphosphorylation of the associated tyrosine kinase, Janus kinase 2 (Jak2), followed by activation of the signal transducer and activator of transcription 5 (Stat5) pathway (16–18), leading to Stat5 translocation to the nucleus and its binding to

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specific promoter sequences (19, 20). PRL was shown to up-regulate ␣2-MG expression via activation of Stat5 in ovarian primary granulosa cells (13, 21) and whole tissue (14, 22). Although the paradigm of PRL signaling through the Stat pathway is well established, very little is known as to how the signal is switched off. Some evidence suggests the involvement of the protein tyrosine phosphatase SHP-2 (SH2-containing protein tyrosine phosphatase-2) in PRL signaling, although this phosphatase acts as a positive rather than negative regulator (23). Recently a new family of SH2-containing proteins, named SOCS (suppressor of cytokine signaling) was discovered and shown to block cytokine signaling (24– 27). Structurally, this family is linked by the presence of a central SH2 domain and a conserved carboxyterminal domain termed the SOCS box. SOCS genes are differentially induced by different cytokines (28– 30). At least eight members are presently grouped in this category (SOCS-1–7 and CIS) (31). SOCS-1 and 3 have been shown to block the activation of gene transcription by PRL and GH, SOCS-1 being a more potent inhibitor (29, 32–34). SOCS-1 has been independently discovered by three groups and named either SOCS-1 (24), JAB (JAK binding protein) (25), or SSI-1 (Stat-inducible Stat inhibitor) (26). The mouse and rat SOCS-1 genes encode proteins of 212 amino acids, whereas the human gene encodes a protein of 211 amino acids. Mouse, rat, and human SOCS-1 proteins share 95–99% amino acid homology (24). SOCS-1 interacts with the catalytic region of Jak kinase, suppresses its tyrosine kinase activity, and thus prevents the phosphorylation of Stat5 (32). It was shown recently to suppress PRL signaling at low levels of expression (32, 33). We have recently found that the rat decidua express SOCS-1. This prompted us to examine the level of SOCS-1 expression in the different decidual cell populations and in PRL-producing and uterine-derived cell lines (35, 36) that either express or do not express the ␣2-MG gene and to examine whether inhibition of SOCS-1 expression with an antisense oligonucleotide can lead to ␣2-MG expression in cells where this gene is usually silenced.

RESULTS

␣2-Macroglobulin mRNA Expression in the Rat Mesometrial and Antimesometrial Decidua and in Two Uterine-Derived Cell Lines The results shown in Fig. 1 confirm our previous results indicating that ␣2-MG mRNA is expressed principally in the mesometrial decidual cells and very little, if any, in the antimesometrial decidual cells. The results also revealed that the ␣2-MG gene is not expressed in the GG-AD cells that were derived from antimesometrial cells (36) but is expressed in UIII cell line. These cells, which originate from endometrial stroma, behave similarly to decidual cells in culture

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Fig. 1. Expression of ␣2-MG in Mesometrial and Antimesometrial Decidual Tissue Total RNA was isolated from day 9 mesometrial (M) and antimesometrial (A) decidual tissue after separation by dissection or from UIII and GG-AD cells cultured as described in Materials and Methods. Touch-down PCR was performed using specific primer sets for ␣2-MG and L-19 as an internal control.

(35). Both cell lines were shown to produce PRL by immunocytochemistry, Western blotting, and RT-PCR (37, 38). Expression of PRL Signaling Components in the Antimesometrial and Mesometrial Decidua and in the Two Uterine-Derived Cell Lines If PRL is responsible for ␣2-MG regulation, any impediment with the normal signaling pathway, such as a deficiency in the expression of an essential transducing component, may prevent the expression of target genes. To test this possibility, we examined the expression of several components known to participate in PRL signaling to the ␣2-MG gene in the two different tissues forming the decidua and in the two uterinederived cell lines. Since maximal expression of ␣2-MG in the mesometrial decidua occurs on days 12–13 of pseudopregnancy, decidual tissue was collected at these days of pseudopregnancy and separated into mesometrial and antimesometrial decidua. Total RNA was subjected to RT-PCR analysis with L19 as an internal control, and we looked at the expression of the positive regulators Jak2, Stat5 (a and b variants), the putative modulator SHP-2, and the negative controller SOCS-1 with that of the target gene. No significant differences in the expression of Jak2 (Fig. 2A) and Stat5 (Fig. 2B) or that of SHP-2 (Fig. 2C) were observed between mesometrial and antimesometrial tissue. In sharp contrast, levels of SOCS-1 expression were vastly different in mesometrial and antimesometrial decidual tissue (Fig. 2D). SOCS-1 mRNA was highly expressed only in the antimesometrial decidua, which does not produce ␣2-MG. To further examine whether the same differential expression of SOCS-1 exists in the two uterine cell

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Fig. 2. PRL Signaling Components in Pseudopregnant Mesometrial (M) and Antimesometrial (A) Decidua Total RNA (1 ␮g) was reverse transcribed and 40 ng of the cDNA were amplified in a touch-down PCR protocol using specific primers for Jak2 and Stat5 (a and b variants), SHP-2, SOCS-1, and L19, which served as an internal control. Lower panels show the densitometric analysis and are mean ⫾ SEM from three independent experiments.

lines, we performed similar experiments in the GG-AD and the UIII cell lines. As shown in Fig. 3, both cell lines express the PRL-R long form (PRL-RL) mRNA. They also express Jak2 and Stat5 (a and b variants) and SHP-2. SOCS-1 was found to be expressed at much lower levels in the ␣2-MG expressing UIII cells than the ␣2-MG silent GG-AD cell line. To examine whether PRL is able to transduce its signal in GG-AD cells that express high levels of SOCS-1, these cells were cultured in the presence of PRL between 0–45 min. Cell extracts were then subjected to immunoprecipitation and Western analysis. Results shown in Figs. 4, 5, and 6 indicate that Jak2 and Stat5 exist as translated products in GG-AD cells (Figs. 4A and 6A). Moreover, PRL is not able to induce Jak2 phosphorylation (Fig. 4B). However, a short 2-min exposure to PRL is sufficient to induce the association of Jak2 with Stat5 (Fig. 5), indicating that Jak/Stat association can occur even between nonphosphorylated Jak2 and Stat5. This association did not lead to Stat5 phosphorylation (Fig. 6B), confirming that Jak2 is inactive, in spite of its transient association with Stat5.

Effect of SOCS-1 Antisense on ␣2-MG mRNA Levels in GG-AD Cells To examine whether SOCS-1 is involved in preventing PRL signaling to the ␣2-MG gene, we challenged the cells with a chimeric phosphorothioate antisense, directed against the 5⬘-terminus of SOCS-1. An irrelevant chimeric phosphorothioate antisense, which was shown to be devoid of any homology with any known gene product, served as a negative control. As shown in Fig. 7, this oligomer prevented the expression of SOCS-1 mRNA in a PRL-independent manner. Moreover, the addition of the SOCS-1 antisense oligonucleotide was able to induce the ␣2-MG gene expression. As shown in Fig. 8, ␣2-MG mRNA became clearly detectable in GG-AD cells after 4 h exposure to the antisense in serum-free medium (lane 2). The ␣2-MG mRNA levels were further increased after 8 h of culture in the presence of serum (Fig. 8, lanes 5 and 6). During this period of time, exogenous PRL had no effect on ␣2-MG gene expression. The lower expression of ␣2-MG in the absence of antisense may be related to decreased endogenous levels of SOCS-1 due to the


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Fig. 3. PRL Signaling Components in UIII and GG-AD Cells Touch-down PCR analysis was performed using specific primer sets for PRL-RL, Jaks (1 and 2), Stat5 (a and b), SHP-2, SOCS-1, and L-19 as an internal control. Total RNA was isolated from UIII and GG-AD cells as described in Materials and Methods.

first period of culture in the absence of serum. However, 24 h later, ␣2-MG expression was observed only in cells transfected with the phosphorothioate antiSOCS-1 and further treated with PRL (Fig. 8, lane 10). This appears to be due to the reduced ability of the GG-AD cells to secrete PRL at this stage of culture (A. Prigent-Tessier and G. Gibori, unpublished) causing the cells to be more responsive to exogenous PRL.

DISCUSSION PRL signal transduction through the activation of the Jak2/Stat5 pathway is well defined. However, the mechanisms by which signaling is prevented are just beginning to be understood. The SOCS protein appears to play an important role in blocking GH- and PRL-induced transactivation of responsive gene promoters (32–34). Whereas in some tissues, little or no SOCS-1 expression is detectable in the absence of stimulation, constitutive expression was observed in others (24). Results of this investigation revealed for the first time that constitutive expression of SOCS-1 can prevent PRL signaling to a PRL-regulated gene in cells producing PRL. The results also revealed that the presence of the PRL-R, Jak2, and Stat5, while being mandatory for PRL signaling, is not a sufficient requirement and that inhibitors of the Jak2/Stat5 signaling pathways in a defined cell population are powerful

molecules that can silence the expression of genes normally up-regulated by PRL. In addition to SOCS-1, SOCS-3 was shown to inhibit the activation of gene transcription by PRL in human mammary cancer cells while SOCS-2 was able to restore PRL signaling (33). Whether SOCS-2 and SOCS-3 play an important role in PRL signal transduction in the decidua needs to be determined. The recent generation of SOCS-1 ⫺/⫺ mice does not allow investigation as to the role of this protein during pregnancy since SOCS-1-deficient mice die before weaning with fatty degeneration of the liver (39). In many cells that do not express the SOCS proteins, cytokines and PRL first activate the Jak/Stat pathway and thereafter stimulate the expression of the SOCS protein that acts to switch off the signaling pathway. In cells that constitutively express SOCS-1, PRL signaling appears to be shut off and PRL-regulated gene expression silenced. This appears to be the reason for which one defined population in the rat decidua expresses the PRL-regulated gene ␣2-MG and another population does not, although both cell types express the PRL-R and are subjected not only to PRL produced by the decidua (38) but also to pituitary PRL and rat placental lactogens produced by the trophoblast. We first thought that the cells that do not express ␣2-MG may lack a critical component in PRL signal transduction. Our results indicate that this is not the case but that Stat5 in these cells is phosphorylated

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Fig. 4. Lack of Jak2 Phosphorylation upon PRL Treatment in GG-AD Cells GG-AD cells were treated with 1 ␮g/ml PRL for the indicated periods of time. Cellular extracts were precipitated with the monoclonal anti-Jak2 antibody and processed for Western blotting analysis as described in Materials and Methods. The blot was first incubated with a monoclonal antibody specific to phosphotyrosine (␣Py) to examine for phosphorylated Jak2 (panel B). The same blot was reprobed, after stripping, with the monoclonal anti-Jak2 antibody (panel A).

neither before PRL treatment nor in response to PRL stimulation, most probably due to the high levels of SOCS-1 expression. Expression of SOCS-1 and ␣2-MG are inversely related, and blocking SOCS-1 expression leads within 4 h to the appearance of ␣2-MG mRNA in GG-AD cells. Thus, the combination of an endogenously generated effector and inhibition of SOCS-1 expression leads to ␣2-MG expression. The constitutive expression of SOCS-1 in the antimesometrial cells and the lack of expression in the mesometrial cells may be of great physiological importance in regard to which cells express ␣2-MG, leading to differential differentiation of decidual cells and allowing for limited trophoblast invasion. Indeed, it is the mesometrial decidua, which lacks SOCS-1 and expresses ␣2-MG, that is the site of trophoblast invasion. These cells are much less differentiated than the antimesometrial cells (1, 2) and remain loosely connected, allowing trophoblast cells to invade without causing massive cell destruction. The limited differentiation of these cells may well be due to ␣2-MG, which binds and prevents the activity of several growth factors involved in cell differentiation (40–48). In addition, the invasive nature of the trophoblast cells is related to the secretion of proteolytic enzymes (49, 50). These trophoblast cells invade without restraint any tissue other than the mesometrial decidua. The abundant secretion of ␣2-MG, a potent protease inhibitor known

to limit trophoblast invasion (50), may be of critical importance for the prevention of mesometrial tissue damage during placentation. The question as to why SOCS-1 is constitutively expressed in one cell type and not the other, causing differential responsiveness to PRL stimulation, remains a subject of further study. Nevertheless, the results of this investigation suggest that the constitutive expression of SOCS, or the lack of it in defined cells of the decidua, may play an important role in the normal development of the placenta.

MATERIALS AND METHODS Chemicals and Biochemicals Tissue culture media M199 and RPMI-1640, antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were obtained from Mediatech (Washington, DC). FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). ExTaq was purchased from Panvera (Madison, WI). The unmodified and phosphorothioate oligonucleotides were obtained from Life Technologies (Gaithersburg, MD). Ovine PRL (APF 10677 C) was provided by the NIDDK (Bethesda, MD). The monoclonal anti-Stat5 (G-2) and anti-p-Tyr (PY99) antibodies and the polyclonal anti-Jak2 (HR-758) antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other reagents were of analytical grade.


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Fig. 5. Coprecipitation of Jak2 and Stat5 after PRL Treatment GG-AD cells were treated with 1 ␮g/ml PRL for the indicated periods of time. Cellular extracts were precipitated with the monoclonal anti-Stat5 antibody separated on 10% denaturing polyacrylamide gel and transferred to nitrocellulose membrane. Blot was incubated simultaneously with the anti-Stat5 and anti-Jak2 antibodies.

Animals and Surgical Procedures Pseudopregnant Holztman rats were obtained from Harlan Sprague Dawley, Inc. (Madison, WI). They were kept under controlled temperature (22–24 C) and light conditions of 14 h light, 10 h dark with free access to standard rat chow and water. Pseudopregnancy was induced by mating females with vasectomized males. The day of vaginal plug was designated as day 1 of pseudopregnancy. Decidualization of the uterine endometrium was induced, under ether anesthesia, on day 5 of pseudopregnancy by scratching the antimesometrial side with a hooked needle. Rats were killed at days 12 and 13 of pseudopregnancy, and uteri were isolated, trimmed of adherent tissue, and washed thoroughly in ice-cold PBS. The mesometrial and antimesometrial decidual tissues were separated as described by Martel et al., (51). Tissue was kept at ⫺80 C until used for RNA isolation.

Cell Culture Both the UIII and the GG-AD cell lines were stably transfected with the PRL-R long form (34, 35) and shown to produce PRL (36, 37) as previously described. The rat endometrial stromal cell line, UIII, is derived from adult Sprague Dawley female rats (34). They express the vimentin filament and have retained several characteristics of uterine stromal cells including progesterone and PRL receptors. These cells also have the ability to differentiate spontaneously in culture, giving rise to large cells that express the desmin intermediate filament and consequently behave as decidual cells. GG-AD are temperature sensitive cells derived from pure rat antimesometrial decidual cells (35). They have retained morphological characteristics of antimesometrial cells: they are polynucleated, large, and have a cytoplasm filled with lipids droplets. They also express the same mRNAs as antimesometrial cells such as activin ␤A and decidual PRL-related protein (dPRP). They were grown in media containing nonessential amino acids (1⫻), antibiotic-antimycotic solution (2⫻), sodium pyruvate (1⫻), D-glucose (0.45%), and FBS (10%). M199 culture medium was used for UIII cells and RPMI-1640 for GG-AD cells. UIII cells were cultured at 37 C, whereas the temperature-sensitive GG-AD cells were first

cultured at 33 C to allow cell growth and then transferred to 39 C before treatment as previously described (35). Culture media were replaced every 48 h and cells were harvested at 70–90% confluence. RNA Isolation and RT-PCR Total RNA was extracted from cells and tissue using guanidium isothiocyanate and phenol in a commercial kit (RNA-NOW, Biogentex, Houston, TX) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed using Advantage RT for PCR (CLONTECH Laboratories, Inc. Palo Alto, CA), and the final volume was adjusted to 100 ␮l. Diluted RT product (3–4 ␮l, representing 30–40 ng of total RNA) was amplified. The reaction mixture consisted of 1⫻PCR buffer (ExTaq buffer, Panvera, Madison, WI), 150 ␮M deoxynucleoside triphosphates, 4.5% dimethylsulfoxide, 20 pmol specific oligonucleotide primers, and 0.8 U ExTaq in a final volume of 40 ␮l. Two sets of amplification cycles were used. In the first five cycles, the annealing and extension temperature of 68 C for 5 min was followed by a denaturation temperature of 93 C for 1 min. In the second set, the annealing temperature of 63 C for 25 sec was followed by a 30-sec extension at 71 C and another 25-sec denaturation at 92 C. Cycle number varied for each of the amplified products and was in the range of 25–35 cycles. The conditions were such that amplification of the product was in the exponential phase, and the assay was linear with respect to the amount of input cDNA. The ribosomal L19 protein was used as internal control to normalize the data. L19 and the specific gene were amplified separately, PCR products were mixed in 1:1 ratio, and 8–20 ␮l of the mix resolved on 2.5% Metaphore agarose gel (FMC Corp. BioProducts, Rockland ME) containing 0.5 ␮g/ml ethidium bromide in 0.75⫻Tris-borateEDTA. The resulting gels were photographed using UV transilluminator and a digital camera (Electrophoresis Documentation and Analysis System 120, Eastman Kodak Co., New Haven, CT). For the detection of Stat5a and Stat5b, a common sense primer 5⬘-GGGCATCACCATTGCTTGGAAG-3⬘ was combined with a specific Stat5a antisense 5⬘-GGAGCTTCTGGCA-

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Fig. 6. Effect of PRL Treatment on Stat5 Phosphorylation in GG-AD Cells Cells were treated with 1 ␮g/ml PRL for the indicated periods of time. Cellular extracts were precipitated with the monoclonal anti-Stat5 antibody, separated on 10% denaturing polyacrylamide gel, and transferred to nitrocellulose membrane. Blot was incubated first with the monoclonal antibody specific to phosphotyrosine (␣Py) to examine for phosphorylated Stat (panel B). After stripping, the same blot was reprobed with the monoclonal anti-Stat5 antibody (panel A). ECL reaction was carried out for 30 sec (A) and for 3 min (B).

Fig. 7. Effect of SOCS-1 Oligonucleotide Antisense on SOCS-1 Gene Expression GG-AD cells were cultured at 33 C to 75% confluence and transferred to 39 C for an additional period of 12 h. They were challenged with lipofectamine coated 250 nM SOCS-1 antisense oligomer for 4 h in Opti-Mem or with the vehicle only. Cells were then washed and complete medium containing either PRL (1 ␮g/ml) or vehicle was added for 20 h. RNA isolation and RT-PCR were as described in Materials and Methods.

GAAGTGAAG-3⬘ or with a specific Stat5b antisense 5⬘-CACGACTAGTATTAACACTTCAC-3⬘ based on the sequences of rat Stat5a (Ref. 52; GenBank accession no. U24175) or 5b (Ref. 53; GenBank accession no. X97541). The sizes of the coamplified cDNA products were 498 and 610 bp for Stat5a and Stat5b, respectively. The other primers were as follows: PRL-RL (54), 5⬘-AAAGTATCTTGTCCAGACTCGCTG-3⬘ and 5⬘-AGCAGTT-

CTTCAGACTTGCCCTT-3⬘ (279 bp cDNA fragment); ␣2-MG (55) 5⬘-GTAATCCTTCTAACTGTTCGGCGA-3⬘ and 5⬘-CCAATGAAGATCGTTTCATACGGA-3⬘ (343 bp cDNA fragment); Jak-1 (Ref. 56; GenBank accession no. AJ000556), 5⬘-CTATGAGCCAGCTGAGTTTCGATC-3⬘ and 5⬘- CATCTCGGACACAGACGCCGTA-3⬘ (275 bp cDNA fragment); Jak-2 (Ref. 57; GenBank accession no. U13396), 5⬘-GTTCTTACCGAAGTGCGTGCGA-3⬘ and 5⬘-GGTAATGGTGTGCATCCGCAGTT-3⬘ (523 bp cDNA fragment); SHP-2 (Ref. 58; GenBank accession no. U09307), 5⬘- CGGGAGTTAAGCAAGCTAGCCG-3⬘ and 5⬘CCTCACACGCATGACGCCATAC-3⬘ (465 bp cDNA fragment); and SOCS-1, 5⬘-GCAGCTCGAAGAGGCAGTCGAA-3⬘ and 5⬘GCTCCCACTCTGATTACCGGCG-3⬘ (273 bp cDNA fragment). No rat SOCS-1 mRNA was ever published in the GenBank database. Thus, we employed BLAST to search for rat homologs to a published mouse sequence (GenBank accession no. U88325). A 13.2-kb rat genomic sequence (GenBank accession no. Z46939) was found that includes, in addition to other genes, the rat SOCS-1 sequence (start site at position 12119, end of last amino acid at position 13155). PCR primers were designed to a piece of this cDNA sequence. A cDNA sequence from a SOCS-1 amplification experiment was sequenced and found to match the rat SOCS-1 sequence. The primers for ribosomal protein L19 were as follows: 5⬘-CTGAAGGTCAAAGGGAATGTG-3⬘ and 5⬘-CGTTCACCTTGATGAGCCCATT-3⬘ (59). Antisense Experiments Chimeric oligonucleotides were designed for the antisense experiments. The SOCS-1 antisense was designed as a 26-bp single-stranded oligonucleotide, covering the ATG start site of the gene with each of the four external nucleo-


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Fig. 8. Effect of SOCS-1 Oligonucleotide Antisense on ␣2-MG Gene Expression GG-AD cells were cultured at 33 C to 75% confluence and transferred to 39 C for an additional period of 12 h. They were then challenged with lipofectamine-coated 250 nM SOCS-1 antisense oligomer or with the irrelevant oligomer for 4 h (lanes 1 and 2) in serum-free medium (Opti-MemI, Life Technologies). Cells were then washed and cultured in RPMI 1640–10% FBS containing either PRL (1 ␮g/ml) or vehicle for another 8 (lanes 3–6) or 24 h (lanes 7–10). RNA isolation and RT-PCR were performed as described in Materials and Methods.

tides on both the 5⬘- and the 3⬘-ends carrying a modified phosphorothioate backbone: 5⬘-CACCTGGTTACGTGCTACCATCCTAC-3⬘. The control antisense oligomer had an identical type of structure, 5⬘-CAGTGCATACGCTGTACGTCATGTAC-3⬘. Cells were grown in RPMI-1640 medium. At 75% confluence, the cultures were transferred to 39 C for 12 h and then washed twice with PBS. The antisense oligomer, precoated with lipofectamine (Life Technologies) at a ratio of 1:26, was added to the cultures at a final concentration of 250 nM in 2 ml Opti-Mem (Life Technologies). After 4 h, cells were washed with PBS and cultured with RPMI-1640–10% FBS supplemented with or without 1 ␮g/ml PRL. Western and Immunoprecipitation Analysis Cells were grown to 80% confluency, washed twice with cold PBS, and lysed at 4 C for 1 h in ice-cold lysis buffer (PBS containing 2% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 2 ␮g/ml of aprotinin, leupeptin, and pepstatin). Cells were scraped, sonicated, and cleared by centrifugation. Protein concentrations were determined using a Protein Assay Dye Reagent kit (Bio-Rad Laboratories, Inc., Hercules, CA). For immunoprecipitation analysis, 800 ␮g protein extracts were incubated overnight at 4 C with monoclonal antiStat5 (G-2) or anti-Jak2 antibodies. Complexes were then precipitated with Protein A/G Sepharose (Santa Cruz Technology, Inc.) and boiled for 5 min in sample buffer: 62.5 mM, Tris-HCl, pH 6.8, 5% ␤-mercaptoethanol, 2% SDS, 20% glycerol, and 0.1% bromophenol blue. Proteins were resolved on 10% denaturing polyacrylamide gels according to the method described by Laemmli (60). After gel electrophoresis, proteins were electrophoretically transferred to nitrocellulose filters (Protran, Schleicher & Schuell, Inc., Keene, NH). The blots were incubated 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) containing 0.1% Tween 20. Blots were washed and incubated overnight at 4 C with the primary antibody (1:2000) and then washed and incubated with a horseradish peroxidaseconjugated antirabbit IgG (1:5000) for 1 h at room temperature. Complexes were visualized using the enhanced chemiluminescence Western blotting detection kit (Western Luminol Reagent; Santa Cruz Biotechnology, Inc.).

Statistics Data were examined by one-way ANOVA, followed by Duncan’s multiple-range test. A level of P ⬍ 0.05 was accepted as statistically significant.

Acknowledgments We are grateful to the NIDDK and the National Hormone and Pituitary Program (NIH) for the oPRL. We thank Dr. He´le`ne Cohen for providing the UIII stromal cell line. We thank Rose Clepper for animal care and Vivian Rogala for preparation of the manuscript.

Received October 15, 1999. Revision received December 22, 1999. Accepted December 28, 1999. Address requests for reprints to: Dr. Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois, 835 South Wolcott Avenue, Chicago, Illinois 606127342. E-mail: [email protected]. This work was supported by NIH Grant HD-12356 (to G.G.) and Ernst Schering Research Foundation (to C.T.).

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Call for Papers The editors of Molecular Endocrinology would like to devote the December 2000 issue to papers that explore the interface between structural biology and molecular endocrinology. With the emergence of structural biology as a discipline, we want to encourage submission of a wide array of manuscripts in this area that can expand our knowledge of the molecular mechanisms underlying hormone action. We are seeking submissions of original research papers for our special issue. Manuscripts will be expected to emphasize novel structures and, when possible, include molecular models. Examples of potential topics include proteomics, structural features of RNA that impact on regulation and processing of mRNA, enzyme catalysis and protein folding, especially that affecting secretory pathways, structural features that govern the thermodynamics of ligand binding, DNA repair mechanisms underlying endocrine cancers, DNA-protein complexes, and chromatin structures that modulate the accessibility of promoter-regulatory regions of genes regulated by hormones. The submission deadline for the special issue is July 1, 2000. Please indicate explicitly in your cover letter that you wish to have the manuscript considered for the special December issue. Any submitted manuscripts that are not published in the December 2000 issue will continue through the normal review process and will be considered for subsequent issues.