Crossing path with smads

Dynamic stability of cells is a function of co-ordination and counterbalance between intracellular signalling events. Therefore, the knowledge of those molecules which form signalling cascades or signalling modules is of prime importance in understanding living processes. Signalling proteins, the component members of different cascades, are the focus of intense interest. Protein phosphorylation dephosphorylation is the prevalent mechanism by which signalling molecules transduce their signals. Therefore, it seems timely to review, in a NATO Advanced Study Institute, those signalling molecules that are involved in the formation of protein kinase modules or more generally in protein complexes like "signallosomes". It is indeed clear that our understanding of such complex structure/function relationships requires a knowledge of the three dimensional structure of the participating protein components. Thus, for example the process of ligand binding, the association of proteins via specific interacting domains, the catalytic mechanism of protein kinases and phosphatases must be described at the atomic level. Likewise an understanding of the temporal and spatial relationship between different signalling molecules within the cellular environment is essential to our comprehension of cellular regulation and, in particular, how normal cells can undergo transformation. Within the context of the Advanced Study Institute, therefore, techniques to study these molecular mechanisms are reviewed. This will comprise: an analysis of structures by X-ray crystallography - the complex situation of a signalling protein within a cell must be looked at, for example, by the manipulation of protein kinase and phosphatase activities within a cell; techniques that can reveal dynamic processes, that is, movement of proteins; and, finally, characterization of proteins at a single molecule level for example in optical traps. Protein kinases is described in much detail as well as the catalytic domains of growth factor receptors. These molecules are located at point of conversion or diversion in regulatory networks. One of the goals of this Advanced Study Institute is to examine the co-ordination of such activities within signalling modules connecting points of junction. Other major players are protein phosphatases which are directed by targeting subunits to specialized locations to keep balance in signalling modules. An exploding area is concerned with lipid metabolism in plasma membranes. Families of lipid kinases like those of the phosphatidylinositol 3-kinases, the phosphatidylinositol 4-kinases, and the phosphatidylinositolphosphate kinases have been described which provide a much more detailed picture of lipid signalling as previously thought. An important aspect is the involvement of these signalling lipids in vesicular transport. It opens the view that these signalling systems participate in a network causing intracellular calcium release, induction of movement, and vesicular transport. It seems, therefore, that an integrated discussion of these diverse phenomena in a NATO Advanced Study Institute may generate a far broader understanding of protein phosphorylation as a major determinant in cellular homeostasis and thus human health and disease

Crossing path with smads

Dynamic stability of cells is a function of co-ordination and counterbalance between intracellular signalling events. Therefore, the knowledge of those molecules which form signalling cascades or signalling modules is of prime importance in understanding living processes. Signalling proteins, the component members of different cascades, are the focus of intense interest. Protein phosphorylation dephosphorylation is the prevalent mechanism by which signalling molecules transduce their signals. Therefore, it seems timely to review, in a NATO Advanced Study Institute, those signalling molecules that are involved in the formation of protein kinase modules or more generally in protein complexes like "signallosomes". It is indeed clear that our understanding of such complex structure/function relationships requires a knowledge of the three dimensional structure of the participating protein components. Thus, for example the process of ligand binding, the association of proteins via specific interacting domains, the catalytic mechanism of protein kinases and phosphatases must be described at the atomic level. Likewise an understanding of the temporal and spatial relationship between different signalling molecules within the cellular environment is essential to our comprehension of cellular regulation and, in particular, how normal cells can undergo transformation. Within the context of the Advanced Study Institute, therefore, techniques to study these molecular mechanisms are reviewed. This will comprise: an analysis of structures by X-ray crystallography - the complex situation of a signalling protein within a cell must be looked at, for example, by the manipulation of protein kinase and phosphatase activities within a cell; techniques that can reveal dynamic processes, that is, movement of proteins; and, finally, characterization of proteins at a single molecule level for example in optical traps. Protein kinases is described in much detail as well as the catalytic domains of growth factor receptors. These molecules are located at point of conversion or diversion in regulatory networks. One of the goals of this Advanced Study Institute is to examine the co-ordination of such activities within signalling modules connecting points of junction. Other major players are protein phosphatases which are directed by targeting subunits to specialized locations to keep balance in signalling modules. An exploding area is concerned with lipid metabolism in plasma membranes. Families of lipid kinases like those of the phosphatidylinositol 3-kinases, the phosphatidylinositol 4-kinases, and the phosphatidylinositolphosphate kinases have been described which provide a much more detailed picture of lipid signalling as previously thought. An important aspect is the involvement of these signalling lipids in vesicular transport. It opens the view that these signalling systems participate in a network causing intracellular calcium release, induction of movement, and vesicular transport. It seems, therefore, that an integrated discussion of these diverse phenomena in a NATO Advanced Study Institute may generate a far broader understanding of protein phosphorylation as a major determinant in cellular homeostasis and thus human health and disease

TGF-β signaling in endometrial cancer

The members of the transforming growth factor-β (TGF-β) superfamily are involved in the regulation of many crucial biological processes including cell proliferation, differentiation, ECM remodelling, metastasis and apoptosis of different cell types. On the other hand, components of the TGF-β signaling cascade are considered classic tumor-suppressors that can play multiple roles in carcinogenesis, acting as tumor suppressors during early stage disease and as tumor promoters at later stages of tumorigenesis. Loss of TGF-β-induced growth inhibition, which is characteristic for many types of cancers, has been associated with disruption and/or dysregulation of the TGF-β pathway, which may facilitate invasion, metastasis, and angiogenesis

Developmental exposure of mice to TCDD elicits a similar uterine phenotype in adult animals as observed in women with endometriosis

Whether environmental toxicants impact an individual woman's risk for developing endometriosis remains uncertain. Although the growth of endometrial glands and stroma at extra-uterine sites is associated with retrograde menstruation, our studies suggest that reduced responsiveness to progesterone may increase the invasive capacity of endometrial tissue in women with endometriosis. Interestingly, our recent studies using isolated human endometrial cells in short-term culture suggest that experimental exposure to the environmental contaminant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) can alter the expression of progesterone receptor isotypes. Compared to adult exposure, toxicant exposure during development can exert a significantly greater biological impact, potentially affecting the incidence of endometriosis in adults. To address this possibility, we exposed mice to TCDD at critical developmental time points and subsequently examined uterine progesterone receptor expression and steroid responsive transforming growth factor-beta2 expression in adult animals. We find that the uterine phenotype of toxicant-exposed mice is markedly similarly to the endometrial phenotype of women with endometriosis

Interferon-γ induces degradation of TβR-I in the human endometrium: Implications for endometriosis

OBJECTIVE: Progesterone action is critical for providing an appropriate endometrial environment for pregnancy. For example, progesterone induces the expression of transforming growth factor- (TGF-2), a signaling molecule involved in down-regulation of matrix metalloproteinases (MMPs).We have demonstrated that progesterone acts through TGF- signaling to limit MMP expression within the inflammatory-like environment of early pregnancy. A recent study demonstrated that interferon-� (IFN-�) related genes are down regulated in leukocytes during the secretory phase, suggesting that progesterone may limit the action of this cytokine in the endometrium. Additional studies have indicated that endometrial tissue from women with endometriosis exhibits a degree of “progesterone resistance” resulting in altered expression of multiple genes during the secretory phase. In the current study, we investigated the potential impact of IFN-� on progesterone-mediated TGF--signaling proteins in the endometrium of women with and without endometriosis. DESIGN: Randomized, controlled laboratory study. MATERIALS AND METHODS: Archived samples of proliferative and secretory phase endometrium from women with and without endometriosis were obtained from the Human Tissue Core of the Women’s Reproductive Health Research Center. Samples were analyzed by standard immunohistochemistry for TGF-1, TGF-2, the type I and type II receptors (TR-I, TR-II) and phosporylated Smad2 (Smad2P). Biopsies of proliferative phase (days 10–12) endometrium was obtained from normal women and from women with a confirmed history of endometriosis. Endometrial stromal and epithelial cells were isolated from biopsies and maintained as co-cultures in the presence of estradiol (E; 1nM), E plus progesterone (EP; 1nM, 500nM) with or without interferon-� (50, 100 or 200 pg/mL). Cells were terminated at 2, 4, 6, 8, 12 and 24 hrs following IFN-� exposure and expression of TR-I, TR-II and Smad2P was examined by Western analysis. RESULTS: Immunohistochemical studies revealed an increased expression of TGF-2, TGF-R-I, TR-II and Smad2P in normal, secretory phase endometrium, but not in similar tissue from endometriosis patients. In vitro treatments with IFN-� induced a dose dependent degradation of TR-I in exposed tissues in the absence of progesterone in all tissues studied. IFN-� effects were not observed prior to 8 hrs, indicating a likely involvement of Smad7 (an inhibitory Smad whose expression is induced 6 hrs after IFN-� treatment). Additionally, Smad2P, an indicator of cellular response to TGF-, was also reduced in both normal and endometriosis endometrium following treatment with IFN-�. CONCLUSION: In women with endometriosis, a diminished response to progesterone increases endometrial MMP expression in response to proinflammatory cytokines. In the current study, we have shown that women with endometriosis have a reduced expression of TGF-2, TR-I and Smad2P and that treatment of normal endometrium with IFN-� results in similar alterations in these molecules. In women with a reduced ability to respond to progesterone, our study suggests that IFN-� action may further reduce the expression of TR-I and prevent the normal regulation of multiple genes including MMPs

Expression and intracellular localization of Smad proteins in human endometrial cancer

The aim of our study was to examine expression of Smad proteins i.e., Smad2, Smad3 and Smad4 both as mRNA and protein as well as their intracellular localization in normal (n=13) and neoplastic (n=42) endometrial tissue specimens using RT-PCR and immunological techniques i.e., Western blot and ELISA. Two uncommon female genital tract tumours, rhabdomyosarcoma of uterine of the cervix and uterine carcinosarcoma were also included. No statistically significant differences were found in the mRNA level of the examined Smad proteins between normal and tumour tissue specimens. Smad2 and Smad3 mRNAs were detected both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix. However, significantly lower Smad2 and Smad4 mRNA level was noted when the depth of myometrial invasion was considered (p<0.05). In endometrial cancer as compared to normal endometrium significantly higher levels of Smad2 and Smad3 proteins, both in cytoplasmic (p=0.002; p=0.0001) and nuclear (p=0.016; p=0.0004) fractions were observed. Both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix Smad2, Smad3 and Smad4 proteins were not detected. Moreover, significantly elevated Smad4 protein level in cytoplasmic fraction was stated when tumour grade and depth of myometrial invasion was undertaken (p<0.05). When intracellular distribution of Smads was considered differences between cytoplasmic and nuclear localization in normal and carcinomatous endometrium was stated. In endometrial cancer decreased number of cases with Smad3 and increased number of cases with Smad4 located in nuclear fraction was found. In conclusion, the disturbances in Smad protein expression and/or differences in their intracellular distribution suggest, that TGF-beta signaling pathway via Smads may be deregulated in endometrial carcinomas

TGF-beta signaling is disrupted in endometrioid-type endometrial carcinomas

OBJECTIVE: Previous studies have demonstrated deregulation of the expression and changes in the intracellular distribution of TGF-beta pathway components in human endometrial cancer (EC). The aim of this study was to assess the relationship between the expression of TGF-beta cascade components, including TGF-beta receptor type I (TGF beta RI) and type II (TGF beta RII), SMAD2, SMAD3, SMAD4, and clinicopathological features--tumor grade, FIGO classification, and depth of myometrial invasion--of type I (endometrioid-type) ECs to give some insight into the role of TGF-beta cascade components in endometrial tumorigenesis. METHODS: The expression of TGF beta RI, TGF beta RII, SMAD2, SMAD3, and SMAD4 was evaluated both at the mRNA and protein level using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively. RESULTS: Infiltrating endometrial carcinomas (less and more than half of the myometrial wall thickness) express significantly higher TGF beta RII protein level compared with non-infiltrating tumors (P = 0.04 and P = 0.01, respectively). Decreased level of SMAD2 and SMAD4 mRNAs was observed in the uterine tumors infiltrating less and more than half of the myometrial wall (P = 0.03 and P = 0.02, respectively) compared with noninfiltrating ECs. Significantly higher SMAD4 protein level in the cytoplasmic fraction of ECs was found when tumor grade and depth of myometrial invasion were considered (P < 0.05). Generally, tumor progression was associated with a decreased number of cases characterized by the presence of SMADs in the nuclear fraction only. CONCLUSION: Our data suggest that disturbances of the TGF beta RII and SMAD4 expression as well as localization of SMADs may be important to the infiltration of the myometrial wall by the type I endometrial carcinomas