Takashi Nagashima, Tetsuo Maruyama, Hiroshi Uchida, Takashi Kajitani, Toru Arase, Masanori Ono, Hideyuki Oda, Maki Kagami, Hirotaka Masuda, Sayaka Nishikawa, Hironori Asada and Yasunori Yoshimura

Progesterone induces decidual transformation of estrogen-primed human endometrial stromal cells (hESCs), critical for implantation and maintenance of pregnancy, through activation of many signaling pathways involving protein kinase A and signal transducer and activator of transcription (STAT)-5. We have previously shown that kinase activation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) kinase is closely associated with decidualization and that SRC is indispensable for maximal decidualization in mice. To address whether SRC kinase activity is essential for decidualization in humans, hESCs were infected with adenoviruses carrying enhanced green fluorescent protein alone (Ad-EGFP), a kinase-inactive dominant-negative mutant (Ad-SRC/K295R), or an inactive autophosphorylation site mutant (Ad-SRC/Y416F). The cells were cultured in the presence of estradiol and progesterone (EP) to induce decidualization and subjected to RT-PCR, immunoblot, and ELISA analyses. Ad-EGFP-infected hESCs exhibited decidual transformation and up-regulation of decidualization markers including IGF binding protein 1 and prolactin in response to 12-d treatment with EP. In contrast, hESCs infected with Ad-SRC/K295R remained morphologically fibroblastoid without production of IGF binding protein 1 and prolactin even after EP treatment. Ad-SRC/Y416F displayed similar but less inhibitory effects on decidualization, compared with Ad-SRC/K295R. During decidualization, STAT5 was phosphorylated on tyrosine 694, a well-known SRC phosphorylation site. Phosphorylation was markedly attenuated by Ad-SRC/K295R but not Ad-EGFP. These results indicate that the SRC-STAT5 pathway is essential for decidualization of hESCs.

DECIDUALIZATION IS THE process by which progestin-induced fibroblastoid stromal cells of estrogen-primed endometrium differentiate into decidual cells. This process is crucial for embryo implantation and maintenance of pregnancy. In the presence of estrogen and progestin, human endometrial stromal cells (hESCs) isolated from human cycling endometrium exhibit morphological and functional changes in vitro that mimic in vivo decidual transformation (1, 2). This in vitro model has enabled a variety of studies on molecular mechanisms underlying decidualization.

To date, cAMP/protein kinase A (PKA) and progestin-mediated signaling pathways have emerged as key cellular events to drive decidual transformation (3). These two signaling pathways cooperatively regulate the activity of decidua-selective transcription factors through their cross talk and convergence, thereby up-regulating decidua-specific genes, eventually leading to terminal differentiation (3). Also, decidualized hESCs produce many bioactive substances, including growth factors and cytokines, whose downstream signaling pathways contribute to decidual transformation in a paracrine/autocrine manner (4, 5, 6). For instance, signal transducer and activator of transcription (STAT)-5, a latent transcription factor activated by numerous cytokines and peptide growth factors, is a candidate signaling molecule thought to regulate decidualization .

We previously reported that kinase activation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) is closely associated with in vitro and in vivo decidualization of hESCs . SRC is a nonreceptor tyrosine kinase that associates with many surface receptors including growth factors and cytokine receptors, becomes activated upon ligand binding, and converts the extracellular stimuli to intracellular signals (12). The kinase activity of SRC is up-regulated by dephosphorylation of its negative regulatory tyrosine residue, tyrosine 527 (corresponding to tyrosine 530 in humans), located at the carboxyl terminus and further enhanced by autophosphorylation of tyrosine 416 (12). Given that many growth factors and cytokines are locally produced from decidual cells (4, 5, 6), it seems reasonable that SRC activation is accompanied by decidualization. However, it remains uncertain whether it is absolutely essential for the process of decidual changes. To address the essential role of SRC in decidualization of hESCs, we previously performed knockdown experiments to abrogate decidual SRC activity using specific inhibitors of SRC family kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) (13); however, these inhibitors unexpectedly promoted decidualization together with paradoxical SRC activation (13). Although we demonstrated that SRC is an indispensable signaling component for maximal decidualization in mice (14), it remains unclear whether SRC and its kinase activity are essential for decidualization in humans.

To clarify this point, we conducted experiments in which adenovirus was used to introduce dominant-negative mutants of SRC into hESCs. We subsequently asked whether the elimination of SRC kinase activity by overexpression of these mutants influenced decidualization of hESCs in vitro. We now provide direct evidence that SRC kinase activation together with STAT5 phosphorylation is required for decidualization of hESCs.

Materials and Method

Antibodies

The mouse monoclonal antibody clone 327, which reacts with both active and inactive SRC, was obtained from Calbiochem (San Diego, CA). BD Living Colors A.v. peptide antibody, which recognizes enhanced green fluorescent protein (EGFP), was purchased from CLONTECH Laboratories (Palo Alto, CA). Phospho-p44/42 MAPK (Thr202/Tyr204) E10 monoclonal antibody, which recognizes the phosphorylated forms of MAPK3/MAPK1, was purchased from New England Biolabs (Beverly, MA). Anti-MAPK3/MAPK1 (total MAPK) antibody was purchased from Upstate Biotechnology (Lake Placid, NY). A monoclonal antibody against β-actin (ACTB) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-STAT5α/STAT5β (total STAT5) polyclonal antibody and anti-phospho-STAT5α/STAT5β (tyrosine 694) polyclonal antibody recognizing the phosphorylated forms of STAT5α/STAT5β were purchased from Cell Signaling Technology (Beverly, MA).

Plasmids and recombinant adenovirus construction

Two pBabe vectors, one encoding a chicken c-SRC kinase-inactive dominant-negative mutant (SRC/K295R) and a second chicken c-SRC inactive autophosphorylation site mutant (SRC/Y416F) were kindly provided by Hidesaburo Hanafusa and Tsuyoshi Akagi (Molecular Oncology, Osaka Bioscience Institute). The recombinant adenovirus vectors carrying SRC/K295R or SRC/Y416F genes were constructed by using the Adeno-X expression system (CLONTECH). Briefly, for the construction of pShuttle SRC/K295R and SRC/Y416F expression vectors, the pBabe-based vectors were digested with BamHI and EcoRI. Then the fragments containing the full-length SRC/K295R or SRC/Y416F cDNAs were blunted with T4 DNA polymerase and ligated with pShuttle vectors that had been digested with NheI and blunt ended. In addition, to construct adenovirus shuttle plasmid pAdeno vectors carrying SRC/K295R or SRC/Y416F, the recombined pShuttle vectors were digested with PI-SceI and I-CeuI, and ligated with pAdeno vectors. The mutated sites and junctions were verified by sequencing and restriction enzyme mapping. The newly recombined pAdeno vectors carrying SRC/K295R or SRC/Y416F were digested with PacI and transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions for the construction of adenovirus vectors. Adenovirus vectors encoding EGFP (Ad-EGFP), SRC/K295R (Ad-SRC/K295R), or SRC/Y416F (Ad-SRC/Y416F) were packaged and propagated in HEK 293 cells. After being purified by cesium chloride gradient centrifugation at 300,000 × g for 3.5 h at 4 C, the adenovirus vectors were dialyzed in PBS overnight at 4 C and stored at −80 C before cell infection in vitro. The viral titer of purified adenovirus was 2.5 × 1010 pfu/ml. About 80% NIH-3T3 cells and 70% hESCs were infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F when the multiplicity of infection reached 50.