Here we describe in detail both the expression of Hoxa-11 in the wild-type mouse uterus and the defects resulting in maternal reproductive failure of Hoxa-11 null female mice. The Hoxa-11 gene is expressed at peak levels in uterine stromal cells during metestrus. Hoxa-11 transcripts were induced beginning on Day 2 of gestation in the stromal cells underlying the uterine epithelium and appeared in the secondary decidual zone between Days 6 and 8 of gestation. At early gestational stages, stromal, decidual, and glandular cell development were deficient in Hoxa-11 null uteri in comparison to wild-type as assessed by histology and immunohistochemical localization of the decidual cell marker epitope, stage-specific embryonic antigen-3 (SSEA-3). Both steroid-induced uterine stromal and glandular cell proliferation as well as oil-induced stromal decidualization after induction of pseudopregnancy were deficient in mutant uteri. Moreover, both Western blotting and immunohistochemistry demonstrated that the burst of glandular leukemia inhibitory factor (LIF) found in normal pregnant uteri at Day 4.5 of gestation was absent in Hoxa-11-deficient uteri. The LIF burst was also not observed in the uteri of bilaterally ovariectomized, hormonally stimulated Hoxa-11 mutants. These results demonstrate that the Hoxa-11 gene is required for normal uterine stromal cell and glandular differentiation during pregnancy, as is the presence of the steroid-induced glandular LIF burst initiating embryo implantation.

BackgroundPulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.MethodsIn vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.ResultsTransgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.ConclusionsBmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.

Mitogen-activated protein (MAP) kinasesRaf-1, pp60O, and p2lm all play important roles in the transfer of signals from the cell surface to the nucleus. We have used the baculovirus/Sf9 insect cell system to elucidate the regulatory relationships between pp60v4vc, p21v-ru ¶ MAP kinase (p44erkl/IaPk), and Raf-1. In Sf9 cells, p44ekl/maPk iS activated by coexpression with either v-Raf or a constitutively activated form of Raf-1 (Raf22W The mechanism of activation of the cytoplasmic serinethreonine kinases p44erkl/mapk and p42erk2/mapk is clearly of interest. In response to extracellular stimuli, p44erkl/maPk and p42erk2/maPk undergo rapid phosphorylation on threonine and tyrosine residues and retardation in gel mobility. Activation of p44erkl/mapk and p42erk2/maPk requires phosphorylation of these proteins on both tyrosine and threonine residues (21, 22). These tyrosine and threonine residues, Thr-183 and Tyr-185 in p42erk2/maPk, are conserved in both isoforms, p44erkl/mapk and p42erk2/mapk. They are the only known cytoplasmic serine-threonine kinases known to be activated by tyrosine phosphorylation. Cytoplasmic MAP kinase activating factors have been identified (23, 24), purified to homogeneity (7, 25, 26), and recently cloned (27). In vitro, activated Raf-1 or v-raf can activate partially purified preparations of MAP kinase activator (14-16).Raf-1 and p44erkl/mapk both appear to be regulated by p21lms and by membrane tyrosine kinases (10,11,28,29). Stimulation of PC12 cells by nerve growth factor or epidermal growth factor activates p44erkl/mapk in these cells. In fact, expression of activated p21lms in PC12 cells is sufficient to activate p44erkl/mapk partially and to cause hyperphosphorylation and retardation in gel mobility of Raf-1 (10, 11). Moreover, expression of a dominant interfering allele of p21c-ms is sufficient to block nerve growth factor-induced p44erkl/mapk activation and hyperphosphorylation of 11).Since both Raf-1 and p44erkl/mapk are regulated by p21V-Ms and by membrane tyrosine kinases, we have used baculovirusencoded p2lv-ras, pp60v-src, p44erkl/mapk, and Raf-1 to examine the effects of each of the activated kinases on the activity of the other. As reported here and by others, p44erkl/mapk is activated by coexpression with either v-Raf or a constitutively activated form of Raf-1 (Raf22W). We report, however, that only a limited increase in the kinase activity of p44erkl/mapk iS seen after coexpression with either Raf-1 or with p21v-ms. The kinase activity of p44erkl/mapk iS greatly enhanced by coexAbbreviations: MAP, mitogen-activated protein; MBP, myelin basic protein.iTo whom reprint requests should be addressed. 5772

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M. (accepted). Development and initial factor validation of the Violence Toward Athletes Questionnaire (VTAQ) in a sample of young athletes. Society and Leisure.