Faculty Bibliography

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-beta-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-beta-catenin signaling with attenuation of the non-canonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.

A reciprocal interaction between the implantation-competent blastocyst and receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. Whereas gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix assisted laser desorption/ionization) mass spectrometry directly on uterine sections, here we provide molecular composition, relative abundance, and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region- and stage-specific manner with the progression of implantation. This application is reliable because patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cytosolic phospholipase A(2alpha) null females that show implantation defects.

Etiology of endometrial cancer (EMC) is not fully understood. Animal models with rapidly and spontaneously developing EMC will help explore mechanisms of cancer initiation and progression. Pten(+/-) mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which approximately 20% progresses to EMC. In addition, tumors develop in other organs, complicating the use of this model to specifically study EMC. Here, we show that conditional deletion of endometrial Pten results in EMC in all female mice as early as age 1 month with myometrial invasion occurring by 3 months. In contrast, conditional deletion of endometrial p53 had no phenotype within this time frame. Whereas mice with endometrial Pten deletion had a life span of approximately 5 months, mice with combined deletion of endometrial Pten and p53 had a shorter life span with an exacerbated disease state. Such rapid development of EMC from homozygous loss of endometrial Pten suggests that this organ is very sensitive to this tumor suppressor gene for tumor development. All lesions at early stages exhibited elevated Cox-2 and phospho-Akt levels, hallmarks of solid tumors. More interestingly, levels of two microRNAs miR-199a(*) and miR-101a that posttranscriptionally inhibit Cox-2 expression were down-regulated in tumors in parallel with Cox-2 up-regulation. This mouse model in which the loxP-Cre system has been used to delete endometrial Pten and/or p53 allows us to study in detail the initiation and progression of EMC. These mouse models have the added advantage because they mimic several features of human EMC.

Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.

Endometriosis is a common gynecological disease that affects approximately 10% of women of childbearing age. It is characterized by endometrial growth outside the uterus and often results in inflamed lesions, pain, and reduced fertility. Although heightened estrogenic activity and/or reduced progesterone responsiveness are considered to be involved in the etiology of endometriosis, neither the extent of their participation nor the underlying mechanisms are clearly understood. Heterogeneous uterine cell types differentially respond to estrogen and progesterone (P(4)). P(4), primarily acting via its nuclear receptor (PR), activates gene transcription and impacts many reproductive processes. Deletion of Fkbp52, an immunophilin cochaperone for PR, results in uterine-specific P(4) resistance in mice, creating an opportunity to study the unique aspects of P(4) signaling in endometriosis. Here we explored the roles of FKBP52 in this disease using Fkbp52(-/-) mice. We found that the loss of FKBP52 encourages the growth of endometriotic lesions with increased inflammation, cell proliferation, and angiogenesis. We also found remarkable down-regulation of FKBP52 in cases of human endometriosis. Our results provide the first evidence corroborated by genetic studies in mice for a potential role of an immunophilin cochaperone in the etiology of human endometriosis. This investigation is highly relevant for clinical application, particularly because P(4) resistance is favorably indicated in endometriosis and other gynecological diseases.

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. Here we report the identification of a mutant, apontic (apt), in which the migratory population expanded and separation from the epithelium was impeded. This phenotype resembled gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimicked loss of function of STAT and its key downstream target, SLBO. APT expression was induced by STAT, which bound directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes.

Macrophage migration inhibitory factor (MIF), a proinflammatory mediator, has been shown to be elevated following heart surgery in adults and may be associated with several postoperative complications, including cardiac and pulmonary dysfunction. In this study, we aimed to measure perioperative plasma MIF, interleukin (IL)-8, and free T4 in 20 children age <4 years undergoing surgical repair of congenital heart lesions with left ventricular volume overload, and to determine whether the response of these mediators determined postoperative outcomes. Plasma samples were obtained preoperatively, immediately on arrival in the pediatric intensive care unit (PICU), and at 12, 24, and 48 h. Patients were continuously monitored in the PICU, and data were recorded daily for therapeutic and monitoring procedures that reflected the invasiveness, intensity, and complexity of care rendered (therapeutic interventional scoring system, TISS). Preoperative plasma MIF, IL-8, and free T4 were not different from age-matched healthy children. However, plasma MIF and IL-8 increased significantly 2 h after completion of cardiopulmonary bypass, and then normalized within 24 h. Peak postoperative levels of MIF (48 +/- 24 ng/mL) and IL-8 (79 +/- 57 pg/mL) correlated significantly with duration of cardiopulmonary bypass. The magnitude of the postoperative increase in plasma MIF was associated with increased number of days required for mechanical ventilation (r = 0.553; P = 0.012), and peak plasma IL-8 correlated significantly with the fraction of inhaled oxygen (FiO(2)) required immediately after surgery (r = 0.510; P = 0.02). Higher circulating MIF levels correlated significantly with increased inotropic support requirements on the second postoperative day, whereas higher postoperative IL-8 levels were associated with higher TISS scores, suggesting increased need for postoperative medical care. These data suggest a potential negative effect of high circulating levels of MIF in the immediate postoperative period on respiratory and cardiovascular functions, and support the development of therapeutic strategies targeting MIF function in this clinical setting.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5'-cap core structure, guanosine(5')triphospho(5')adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5')tetraphospho(5')adenosine (GppppA), that is formed by the transfer of the 5'-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the gamma-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.