Bidirectional signaling between the cytoskeleton and integrins

Clustering of integrins into focal adhesions and focal complexes is regulated by the actin cytoskeleton. In turn, actin dynamics are governed by Rho family GTPases. Integrin-mediated adhesion activates these GTPases, triggering assembly of filopodia, lamellipodia and stress fibers. In the past few years, signaling pathways have begun to be identified that promote focal adhesion disassembly and integrin dispersal. Many of these pathways result in decreased myosin-mediated cell contractility.     

Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium

The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17beta-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17beta-estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17beta-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17beta-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17beta-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.     

Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis

Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.     

Cross-talk between estrogen and leptin signaling in the hypothalamus

Obesity, characterized by enhanced food intake (hyperphagia) and reduced energy expenditure that results in the accumulation of body fat, is a major risk factor for various diseases, including diabetes, cardiovascular disease, and cancer. In the United States, more than half of adults are overweight, and this number continues to increase. The adipocyte-secreted hormone leptin and its downstream signaling mediators play crucial roles in the regulation of energy balance. Leptin decreases feeding while increasing energy expenditure and permitting energy-intensive neuroendocrine processes, such as reproduction. Thus, leptin also modulates the neuroendocrine reproductive axis. The gonadal steroid hormone estrogen plays a central role in the regulation of reproduction and also contributes to the regulation of energy balance. Estrogen deficiency promotes feeding and weight gain, and estrogen facilitates, and to some extent mimics, some actions of leptin. In this review, we examine the functions of estrogen and leptin in the brain, with a focus on mechanisms by which leptin and estrogen cooperate in the regulation of energy homeostasis.     

Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae

The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions. Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae. In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling. Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p. Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p. Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein). Therefore, we analyzed additional SCF mutants for chromatin assembly defects. Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly. Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p. However, rsp5CA and Deltaubc7 mutations had opposite genetic effects on apc5CA and cdc34-2 phenotypes. Therefore, the antagonistic interplay between Deltaubc7 and rsp5CA, with respect to cdc34-2 and apc5CA, indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p.     

Modeling interaction between gp120 HIV protein and CCR5 receptor

The study of the process of HIV entry into the host cell and the creation of biomimetic nanosystems that are able to selectively bind viral particles and proteins is a high priority research area for the development of novel diagnostic tools and treatment of HIV infection. Recently, we described multilayer nanoparticles (nanotraps) with heparin surface and cationic peptides comprising the N‐terminal tail (Nt) and the second extracellular loop (ECL2) of CCR5 receptor, which could bind with high affinity some inflammatory chemokines, in particular, Rantes. Because of the similarity of the binding determinants in CCR5 structure, both for chemokines and gp120 HIV protein, here we expand this approach to the study of the interactions of these biomimetic nanosystems and their components with the peptide representing the V3 loop of the activated form of gp120. According to surface plasmon resonance results, a conformational rearrangement is involved in the process of V3 and CCR5 fragments binding. As in the case of Rantes, ECL2 peptide showed much higher affinity to V3 peptide than Nt (K_D = 3.72 × 10^?8 and 1.10 × 10^?6 M, respectively). Heparin‐covered nanoparticles bearing CCR5 peptides effectively bound V3 as well. The presence of both heparin and the peptides in the structure of the nanotraps was shown to be crucial for the interaction with the V3 loop. Thus, short cationic peptides ECL2 and Nt proved to be excellent candidates for the design of CCR5 receptor mimetics.     

The role of the interaction between signaling protein domains and of the complexes of signaling proteins in apoptosis initiation

The data of recent years on apoptosis were revisited to demonstrate that the functioning of signaling proteins during apoptosis depends on their localization on mitochondria or in the cytosol. The major effect of signaling proteins depends on the number of pro-and antiapoptotic domains in their structure, which is observed after cleavage, oligomerization, and complexing with other proteins. The structure of known signaling proteins was analyzed. The effect of complexing with phosphatases and 14-3-3 proteins was demonstrated by the example of Bad protein. Detailed data on the proapoptotic factors and their inhibitors affecting caspase activation and released from mitochondria with cytochrome c are given.     

Functional interaction between the pp71 protein of human cytomegalovirus and the PML-interacting protein human Daxx

The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.     

Detection and synthesis of a progestagen-dependent protein in human endometrium

Immunologiocal and biochemical methods were employed to demonstrate the presence of progestagen-dependent proteins in human endometrium. Cytosols were prepared from proliferative and secretory phase endometria of cycling women, from decidua and decidua-rich tissues of women in early pregnancy and from decidua of tubal pregnancy. Antisera were raised in rabbits against the antigens of decidua of tubal pregnancy and decidua-rich tissues. Immunoelectrophoresis, Ouchterlony's immunodiffusion test and polyacrylamide gel electrophoresis using native gels revealed 2 antigenic proteins, designated antigens A and B, in secretory endometria, decidua-rich tissues, decidua, and in decidua of tubal pregnancy. However, only 1 antigenic protein was detected by SDS-gel electrophoresis: antigens A and B may therefore be two different proteins or two forms of a single protein. The antigens could not be detected in non-pregnancy sera or in term placentae. Double isotopic labelling (incubation of tissues with [3H]- and [14C]leucine) followed by protein fractionation methods were used to compare the in-vitro synthesis rates of antigens in poroliferative tissues with those in decidua or secretory endometria. The rate of synthesis of antigens A and B was markedly higher in the decidua and secretory endometria than in the proliferative endometria. We conclude, therefore, that during progestagen-dependent transformation of proliferative phase endometria into secretory endometria and decidua in women, there is a selective stimulation of at least one species of pregnancy-associated protein.     

Specific regulation by steroid hormones of protein kinases in the endometrium. 1. Alteration by estrogen and progesterone in levels of protein kinases in rabbit endometrium

The alteration in activities of multiple protein kinases has been studied in the endometrium of a rabbit treated with estrogen and progesterone. The administration of estrogen or progesterone to the castrated rabbit resulted in a remarkable increase of total activity in the cytosol fraction of the endometrium. The administration of estrogen caused an increase of type I adenosine-3',5'-monophosphate-dependent (cAMP-dependent) protein kinase and a slight decrease of type II cAMP-dependent protein kinase. In contrast, the treatment with progesterone after priming administration of estrogen brought about an increase of type II cAMP-dependent protein kinase and a decrease of type I cAMP-dependent protein kinase. Therefore, the activity ratio of type II to type I decreased by estrogen and increased by progesterone. The simultaneous administration of cycloheximide abolished the stimulatory effect of respective hormones on the level of each protein kinase. The activity profile of protein kinases on DEAE-cellulose column after ovulation caused by the administration of human chorionic gonadotropin to a non-castrated rabbit was similar to that of the rabbit treated with progesterone. The results presented demonstrate the specific regulation by the steroid hormones of de novo synthesis of protein kinases in the target organ.     

DNA and nuclear protein measurement in isolated nuclei of human endometrium

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.