Endothelial tight junction proteins
The endothelium is situated at the inner side of all kinds of vessels and comprises of a monolayer of endothelial cells. Inter-endothelial junctions comprise junctional complexes, such as adherens junctions (AJ), tight junctions (TJ) and gap junctions (GJ) that play essential roles in tissue integrity, barrier function and intercellular communication respectively. These junctional complexes are related to those found at epithelial junctions with notable changes in terms of certain molecules and structure.
Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. Dormant endothelium may be exposed to stimuli provoking leukocyte extravasation at seditious sites and propagating angiogenesis. Both activities have an intense impact on endothelial cell-cell junctions.
Tight junctions aid the major functional objective of establishing a barrier inside the membrane, by controlling paracellular permeability and sustaining cell polarity. They achieve this by constricting apical or basolateral transmembrane diffusion of lipids and they have been suggested to contribute in regulating proliferation and differentiation of epithelial cells. However, the components that are involved and the signal routes concerned are unknown (Mitic & Anderson 1998).
Tight junctions are made up of integral membrane proteins claudins, occludin, tricellulin, junctional adhesion molecules (JAMs), including many peripheral membrane proteins such as the scaffold PDZ- domain proteins. This review will however, focus on ZO-1 and ZONAB.
Histology of endothelia junctions
The junctional structures situated at the endothelial intercellular fissure are related to those located at the epithelium; however, their formation is more inconsistent and in most vascular beds their topology is less constrained than in epithelial cells. Adherens junctions, tight junctions and gap junctions are in most cases intermingled and create a complex zonular system with disparities in depth and thickness of the sub-membrane plate associated with the junctional structure (Franke et al. 1988; Rhodin 1974). In contrast to epithelial cells, GJs are often found close to the luminal surface. Hence, the term “Apical junction” used to jointly describe epithelial TJ and AJ may not be applied to the endothelium. The endothelium forms the vascular barrier with controlled permeability properties between the blood and the underlying tissues.
Tight junctions exhibit considerable inconsistency among different segments of the vascular tree (Franke et al. 1988). This disparity composes a major evidence of vascular bed differentiation of endothelial cells and has a strong impact on vascular permeability and leukocyte extravasation. Variations concern the complexity degree of the occluding strands as well as tight junction composition.
Large Artery endothelial cells, which are exposed to high flow rates, display a well-developed system of tight junctions. Within the microvasculature, tight junctions are less complex in capillaries than in arterioles, and even less in venules. It is important to mention that, post-capillary venules are the primary site of leukocyte extravasation, and accordingly, they display a high content of permeability mediator receptors, such as those for histamine, serotonin and bradykinin. On the other hand, blood brain barrier (BBB) and the blood retinal barrier (BRB) are predominantly rich in Tight Junctions and endothelial tight junctions have been principally studied in these sites.
Endothelial intercellular realms differ from those of epithelial cells by the absence of desmosomes (Franke et al. 1988). The transitional filaments, comprised in the endothelium by vimentin molecules, are poorly connected to cell-cell contacts. However, contrary to the situation in epithelia, the vimentin filaments may be associated to endothelial adherens junctions in junctional structures similar to desmosomes, called complexus adherens.
It must be emphasized that interendothelial junctions are vibrant structures, subjected to multiple regulations. Moreover, leukocytes extravasate majorly in postcapillary venules either through transcellular or paracellular methods. Extravasation via the intercellular junction is a rapid and controlled process, through which the leukocyte is squeezed in the fissure (diapedesis), followed by rapid junction reformation.
ZO-1 is a protein located on the cytoplasmic membrane plate of intercellular tight junctions and is engaged in transducing signals at cell-to-cell junctions. ZO-1 links tight junction transmembrane proteins to a cytoplasmic plaque and the actin-based cytoskeleton (Aijaz et al. 2006; Tsukita et al. 2001). In epithelial cells, ZO-1 interrelates with the transcription factor ZONAB to regulate cells proliferation in a cell density related manner (Balda & Matter 2000); however, the functions of ZO-1 and ZONAB in endothelial cells are still not clearly understood.
Unpublished work shows that downregulation of ZO-1 in endothelial cells stimulates redistribution of two transmembrane proteins; claudin-5 and JAM-A, and radical changes in the cytoskeleton affecting the localization of mechanosensor proteins and VE-cadherin role in the control of cell-cell tension.
These observations imply that one function of ZO-1 in endothelial cells is to coordinate components of the tight junction and associate them to the cortical cytoskeleton. However, it is unfamiliar whether the ZO-1 associated transcription factor ZONAB is linked to such ZO-1 effects.
Despite the fact that, ZO-1 explicitly associates with epithelial tight junctions (Stevenson et al. 1986), it has been observed that the protein appears in the nucleus in the process of proliferation (Gottardi et al. 1996). While the functional impact of the nuclear localization is currently not clear, studies reveal that these discrete subcellular distributions of ZO-1 are exquisitely sensitive to the state of cell-to-cell contact.
ZO-1 plays a major role of restraining ZONAB and regulates its accumulation in the nucleus through cytoplasmic sequestration. MDCK cells found in the epithelium exhibit two forms of this Y-box transcription factor (ZONAB) i.e. ZONAB -A and ZONAB -B which vary in a 68-amino acid supplement. Both categories of ZONAB bind to ZO-1 and link with intercellular junctions (Balda & Matter 2000).
ZONAB was initially designated in canine kidney epithelial cells (MDCK) and is a Y-box transcription factor. Y-box transcription factors are multipurpose control mechanisms of gene expression and studies suggest that they play a common role in enhancing proliferation (Bargou et al. 1997). ZONAB is one of the tight junction-associated dual localization protein: it localizes to junctions where it attaches to the SH3 surface of the adaptor protein ZO-1, and to the nucleus where it regulates transcription.
The distribution of ZONAB is controlled by the cell density as it localizes to both junctions and nuclei in low density, proliferating cells, and becomes constrained to the cytoplasm in high density cells (Balda & Matter, 2000). This distribution is also exhibited in its transcription activity, as ZONAB is transcriptionally vigorous in proliferating cells but inactive in non-proliferating cells. In the MDCK cells, ZONAB is necessary for normal rates of proliferation and controls G1/S phase transition (Balda et al. 2003).
ZONAB affects cell cycle development by two distinct processes: it controls the nuclear accumulation of CDK4 through a direct interaction and controls manifestation of genes encoding cell cycle regulators for example, PCNA and cyclin D1 (Balda et al. 2003; Sourisseau et al. 2006 ).
In 3D principles of MDCK cells, regular ZO-1 and ZONAB processes are necessary for epithelial cyst formation, implying that the Y-box transcription factor also controls epithelial differentiation (Sourisseau et al. 2006). Since ZO-1 and ZONAB can also relate with other types of intercellular junctions, for instance the gap junctions, in cells that lack tight junctions, it is possible that ZO-1 or ZONAB signaling is also of useful significance in other cell types other than epithelia (Ciolofan et al. 2006; Giepmans & Moolenaar 1998).
Aims of the study
The aim of the study is to understand the functional consequences of downregulation of ZONAB in endothelial cells, and whether and how ZONAB cross-talks with other junctional components to regulate endothelial cell migration, proliferation and angiogenesis. Currently, we are looking at similarities and differences between the phenotype of downregulation of ZO-1 or ZONAB by RNA interference. Changes in expression and localization of a given protein are analysed using specific antibodies for immunoblots and immunofluorescence.
It is observed that downregulation of ZO-1 or ZONAB resulted in similar redistribution of actin and vinculin from cell-cell junctions to stress fibers and focal adhesions, respectively. However, the localization of transmembrane proteins such as Claudin-5 and JAM-A is affected by downregulation of ZO-1 rather than by downregulation of ZONAB. The localization of the polarity protein PAR-3 is changed in both conditions.
Additionally, downregulation of ZONAB causes changes in ZO-1 by immunofluorescence that needs to be tested for expression by immunoblots. Next, we will characterize other transmembrane proteins (e.g. MD3 and claudin-1), polarity proteins (PKCzeta), Rho regulators and mechanotransducers such as PAK2, Zyxin and YAP.
ZONAB is a DNA and RNA binding factor that it is involved in transcription (e.g. cyclin D1 and PCNA) in the nucleus and translation (e.g. cell cycle inhibitor p21) in the cytosol. Thus, we are also trying to identify new genes regulated. We have identified that expression of fibronectin is regulated by ZONAB. We are evaluating whether the changes in protein expression of fibronectin are due to ZONAB role on transcription or translation, using actinomicin D to inhibit transcription or cyclohexidimide to inhibit translation. Additionally, we are validating new genes identified by cDNA array analysis of endothelial cells with downregulation of ZONAB.
The tight junction localizing protein ZO-1 symptomatically forms a continuous band around the apices of well-differentiated, confluent, polarized epithelial cells in culture. However, under nonconfluent conditions, endogenous ZO-1 can localize to the nucleus in addition to the border of cell-cell contact.
ZONAB manifestation tends to be high in proliferating but low in growth-impeded MDCK cells, implying that high manifestation levels might be a necessity for cell proliferation (Balda & Matter 2000).
ZONAB confines in the nucleus as well as tight junctions in proliferating cells, however, it is not noticeable in the nucleus of nonproliferating high density cells (Balda & Matter 2000), proposing that accumulation of ZONAB in the nucleus might be necessary for efficient proliferation.
ZO-1 quantities are low in proliferating cells and they rise with cell density, and overexpression of ZO-1 hinders accumulation of ZONAB in the nucleus (Balda & Matter 2000); hence, ZO-1 may control proliferation by inhibiting ZONAB from accumulating in the nucleus. Overexpression of ZO-1 in low density cells triggers a redistribution of ZONAB from the nucleus to the cytoplasm and reduced proliferation.
CDK4 is a major regulator of G1/s transition (Sherr 2000; Malumbres & Barbacid 2001). Thus, ZONAB could control proliferation by regulating the process or the localization of CDK4. Since ZONAB binds CDK4, the nuclear pools of the two proteins may diminish in a parallel manner.
Symplekin is combined with ZONAB in the nucleus; hence, it could be argued that Symplekin modulates the transcription activity of ZONAB. Increased expression of Symplekin results in stimulation of the transcriptional suppressor ZONAB. However, it is also noted that Symplekin is absent in endothelial cells (Keon et al. 1996).
ZONAB controls cell cycle entry. ZO-1 overexpression results in a reduction in DNA synthesis, implying that entry into S-phase was distressed.
These experiments will allow understanding the role of ZO-1 and ZONAB in endothelial cells. Depending on the results, we plan to test how these two proteins are involved in endothelial stress conditions such as shear stress and high glucose.
The collaboration of ZO-1 with tight junctions can only be significant for the stabilization of ZO-1, as opposed to attaching ZO-1 to the plasma membrane so as to constrain nuclear accumulation of related proteins. This is supported by the opinion that a truncated protein comprising only the HA-tagged SH3 domain accumulated in the Cytosol, but was adequate to decrease proliferation and nuclear accumulation of ZONAB (unpublished data).
ZONAB and ZO-1 control proliferation and the ultimate cell density of MDCK cells. Explanations that ZO-1 accumulates with increasing cell density, and overexpression of ZO-1 in transfected cells lowers the final density proposes a pattern in which ZO-1 serves as a measure for cell density whereby, on reaching the threshold level, provokes growth impediment by cytoplasmic sequestration of ZONAB and the related cell cycle kinase CDK4. It will be essential to control how the ZO-1 or ZONAB pathway associates with the other signaling methods that affect proliferation.
Vascular endothelial stress induces dysfunctions that have been implicated in many diseases such as diabetes and diabetic retinopathy. Therefore, characterization of the role of tight junction molecules in different endothelial cell behavior and functions will help us to understand the molecular mechanisms of disease pathogenesis and these findings may be implicated in prognosis and possibly to develop new treatment strategies.
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