Role of rac1 protein in the acquisition and mainteinance of epithelial cell polarity

Abstract: The acquisition of cell polarity, which includes the establishment of the tight junction barrier, the polarized assembly of the cytoskeleton and the appropriate organization of membrane traffic, requires external cues, that in epithelial cells are represented by the interaction of cells with their neighbors and with the extracellular matrix. The Rho family of small GTPases, whose prototypes are RhoA, Rac1 and Cdc42, regulates many biological processes including cell cycle progression, apoptosis, migration and intercellular adhesion. Rho-GTPases and their effectors are also key regulators of microfilament and microtubule dynamics and, consequently, are crucially involved in polarity signaling. Unraveling how signaling transduced via Rac1 are translated into oriented distribution of molecules in epithelial cells is a central issue to fully understand the processes of acquisition/maintenance of cell polarity. In order to contribute to the understanding of the molecular basis of this complex issue in the present study we have focused our attention on the analysis of the role of Rac1 protein in the acquisition and mantainance of the polarized phenotype in the FRT, rat thyroid epithelial cell line. The FRT cells were chosen as a model system since they exhibit a fully polarized epithelial phenotype, manifest high transepithelial electrical resistance and express apical and basolateral marker proteins. They also form organized tridimensional follicular structures in suspension culture. The Rac1 subcellular distribution in FRT cells was initially investigated. Subsequentely, to analyze the role of Rac1 in the control of cell polarization in the FRT cells, the cultures were treated with NSC23766, a molecule that does not allow Rac1 specific GEFs, such as Tiam1 and Trio, to bind to Rac1 and therefore acts as an inibitor of Rac1 activation. Several aspects including directional migration, TER acquisition, cell aggregation and formation of polarized follicles were investigated and found to be affected by the pharmacological inhibition of Rac1 activity. Moreover in this type of experiment, changes in subcellular localization of the Rac1 molecule were analyzed in parallel with those regarding E-cadherin. This experimental appoach allowed us to establish that Rac1 is a major regulator of the polarization process in FRT cells. The data presented also suggest that, in addition to the control of cell-cell adhesion, Rac1 may be involved also in the control of the Golgi apparatus integrity and therefore in the polarized intracellular traffic of proteins. The role of integrin signaling in the acquisition/maintenance of cell polarity has been studied to some extent in polarized epithelial cells in culture. For example in the FRT-β1B cells, derived from FRT parental cells after transfection of the dominant-negative β1B integrin, an impairment of the polarized phenotype have been described. The observation that these cells manifest properties similar to those evidenced in FRT cells where Rac1 is inhibited prompted us to test the hypothesis that this may be due to a reduced Rac1 activity. Subclones derived from FRT-β1B cells were obtained that stably express a Rac1 construct that is constitutively activated upon 4-OH-tamoxifen treatment. However it was not possibile to rescue the normal phenotype in these cells by this experimental approach. Furthermore also FRT parental cells expressing the same construct appeared to be hampered in the expression of certain properties of the polarized epithelium, indicating that sustained but not regulated acivation of Rac1 impairs the acquisition of cell polarity. In conclusion this report combines the interest in defining the role of Rac1 in a model of thyroid derived-epithelium, in which the functional properties of this small Rho GTPase heve not been investigated yet, to novel experimental approaches, such as the use of the Rac1-GEFs-interaction inhibitor and a novel conditional expression vector of Rac1 protein

Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts.

Keloids are benign skin tumors and are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are characterized by formation of excess scar tissue beyond the boundaries of the wound. Keloids are often confused with hypertrophic scars because of an apparent lack of morphologic differences. The molecular distinction between scars and keloid is still controversial and, until today, there is no appropriate treatment yet for keloid disease. In this study, we have found, for the first time, p53 mutations in both hypertrophic scar and keloids fibroblasts from cultured cells to various extents. Since p53 plays a central role in the DNA damage response by inducing cell cycle arrest and/or apoptotic cell death, we also set up time course experiments making cell cultures at different times to investigate the phenomenon of apoptosis and its involvement in the process of pathological scarring in both hypertrophic scars and keloids. The extent of apoptosis in this study was investigated by DNA fragmentation and MTT assays, propidium iodide staining, p53 expression, and subcellular distribution. Moreover, the correlation of apoptosis and ROS levels in keloid and hypertrophic scars fibroblasts was assessed. Understanding the molecular mechanisms that determine the regulation of apoptosis during wound healing might allow us to therapeutically modulate these pathways so that apoptotic cell death is reactivated in dysregulated and hypertrophi

Rac activity regulates polarity of thyroid epithelial cells

Thyroid derived FRT cells express in culture the polarized epithelial phenotype. FRT-β8i cells, in which the signal transduction from the β1A integrin is impaired by the expression of the β1B variant, have a partial polarization defect. We tested the hypothesis that this is due to a lack of activity of the Rac GTPase. By confocal microscopy the endogenous Rac, as well as Rac-GFP, is present in the cytosol of FRT cells but also on the plasma membrane. In confluent monolayers on filters Rac forms a ring that is associated to the tight junction. The Rac-specific inhibitor NSC23766 inhibits FRT cell migration, reduces the amount of Rac on the plasma membrane and prevents the full acquisition of transepithelial resistance (TER). This effect is reversible and is not observed if the drug is added when the monolayer has acquired its full TER. The Rac inhibitor also, in part, impairs the formation of polarized cysts in suspension culture. The phenotype of the NSC23766-treated FRT cells is fully reminiscent of that of FRT-β8i suggesting that Rac signaling regulates the polarized phenotype. We are now trying to correct the FRT-β8i phenotype by using an inducible ER-Rac(QL)

Differential p63 and p53 expression in human keloid fibroblasts and hypertrophic scar fibroblasts.

The p63 gene belongs to the p53 gene family and encodes for sequence-specific transcription factors. p63 has been characterized primarily in the context of epidermis where is implicated in the establishment of keratinocyte cell fate and in maintenance of epithelial self-renewal. DeltaNp63 isoform has been showed to be involved in several kinds of human tumors of epidermal origin, even nonmalignant, for the neoplastic and proliferative potential. Here, we report the differential expression and the cellular localization of the DeltaNp63 isoform in fibroblasts isolated from human keloids and hypertrophic scars compared to normal skin. Differently from hypertrophic scar, our results show that DeltaNp63 has a nuclear localization and is overexpressed only in keloid fibroblasts, suggesting an essential role of DeltaNp63 in vivo in human keloids. Consistent with our results, we hypothesize that DeltaNp63 overexpression may be oncogenic because of its ability to block the activity of p53 since p53 is underexpressed in fibroblasts from keloids

Membrane association of Rac and E-cadherin in FRT thyroid epithelial cells.

We are interested in determining the molecular mechanism by which Rac affects the expression of the polarized phenotype in FRT thyroid epithelial cells. To this aim an inducible, constitutively-active form of Rac, ER-Rac(QL), and the inducible, dominant-negative ER-Rac(N17) were stably expressed in FRT cells. By immunofluorescence analysis and cell fractionation we determined that upon tamoxifen treatment of FRT clones expressing ER-Rac(QL), the protein moves from the cytosol to the plasma membrane. The same is true for the dominant-negative ER-Rac(N17) after tamoxifen treatment. The bulk of endogenous Rac is also localized on the plasma membrane of wild-type FRT cells. Treatment with the specific Rac inhibitor NSC23766, removes endogenous Rac from the plasma membrane. Strikingly, E-cadherin is correspondingly removed from the membrane, likely by endocytosis. Chelation of calcium in the culture medium, in a Ca++ switch assay, also causes internalization of E-cadherin from the plasma membrane and the partial removal of Rac. Intracellular E-cadherin and Rac do not colocalize. The coordinate regulation of the association of both proteins to the plasma membrane is under investigation

A constitutively active Rac impairs the acquisition of epithelial cell polarity.

We demonstrated that treatment of FRT thyroid epithelial cells with a Rac-specific inhibitor causes an impairment in the acquisition of polarity. FRT-β8i cells, in which the signal transduction from the β1A integrin is impaired, manifest a similar defect. We tested here the hypothesis that an active Rac might correct the FRT-β8i polarity defect. FRT-β8i cells that stably expressed an inducible, constitutively active Rac, ER-Rac(QL), were obtained. Upon tamoxifen treatment the ER-Rac(QL) protein became active, localized at the plasma membrane and in confluent cells it was mostly found on the lateral plasmamembrane, at sites of cell-cell contact. In these cultured cells cytokinesis was progressively impaired. Furthermore, activation of ER-Rac(QL) interfered with the acquisition of transepithelial resistance by confluent monolayers on filters, impaired cyst formation by cells in suspension culture and reduced the wound healing efficiency in a scratch test. Similar results were obtained with wild-type FRT cells expressing the same ER-Rac(QL). We conclude that a constitutively active Rac does not promote, but rather hampers, the acquisition of cell polarity in epithelial cells

Binding of Carbonic Anhydrase IX to 45S rDNA Genes Is Prevented by Exportin-1 in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45S rDNA gene promoters