Tomonori Hirose

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM)

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning‐defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR‐3 and PAR‐6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR‐3 associates with the tight‐junction‐associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin‐1, ‐4 or ‐5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell–cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR‐3 localization at intercellular junctions and delays ASIP/PAR‐3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR‐3–aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.

Mammalian Lgl Forms a Protein Complex with PAR-6 and aPKC Independently of PAR-3 to Regulate Epithelial Cell Polarity

BACKGROUND Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. RESULTS We show that mammalian Lgl competes for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with the previous observation that the complex containing PAR-3 promotes it. CONCLUSIONS These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity.

PAR‐6 regulates aPKC activity in a novel way and mediates cell‐cell contact‐induced formation of the epithelial junctional complex

Background PAR‐6, aPKC and PAR‐3 are polarity proteins that co‐operate in the establishment of cell polarity in Caenorhabditis elegans and Drosophila embryos. We have recently shown that mammalian aPKC is required for the formation of the epithelia‐specific cell‐cell junctional structure. We have also revealed that a mammalian PAR‐6 forms a ternary complex with aPKC and ASIP/PAR‐3, and localizes at the most apical end of the junctional complex in epithelial cells.

Spatial regulation of VEGF receptor endocytosis in angiogenesis

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

Regulated protein–protein interaction between aPKC and PAR-3 plays an essential role in the polarization of epithelial cells

Background: Recent studies have revealed that aPKC (atypical protein kinase C), PAR‐3 and PAR‐6 play indispensable roles in the regulation of various cell polarization events, from worms to mammals, suggesting that they comprise an evolutionarily conserved protein machinery which is essential for cell polarization. The three proteins interact with each other to form a ternary complex and thus mutually regulate their functionality and localization. Here, we investigated the biochemical nature of the aPKC–PAR‐3 interaction in detail to clarify its functional importance in cell polarity.

PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6β and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 andα 4/β1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.

ASPP2 Regulates Epithelial Cell Polarity through the PAR Complex

The PAR complex, consisting of the evolutionarily conserved PAR-3, PAR-6, and aPKC, regulates cell polarity in many cell types, including epithelial cells [1-4]. Consistently, genetic manipulation of its components affects tissue integrity in multiple biological systems [5-9]. However, the regulatory mechanisms of the PAR complex remain obscure. We report here that apoptosis-stimulating protein of p53 (ASPP2 or TP53BP2), which binds to the tumor suppressor p53 and stimulates its proapoptotic function [10-12], positively regulates epithelial cell polarity by associating with the PAR complex. ASPP2 interacts and colocalizes with PAR-3 at apical cell-cell junctions in the polarized epithelial cells. Depletion of ASPP2 in epithelial cells causes defects in cell polarity, such as the formation of tight junctions and the maintenance and development of apical membrane domains. Moreover, depletion of ASPP2 causes a defect in PAR-3 localization, as well as vice versa. Furthermore, disturbance in the interaction between ASPP2 and PAR-3 causes defects in cell polarity. We conclude that ASPP2 regulates epithelial cell polarity in cooperation with PAR-3 to form an active PAR complex. Our results, taken together with the known functions of ASPP2, suggest a close relationship between cell polarity and other cell regulatory mechanisms mediated by ASPP2.

Expression of Toll-like receptor 9 in renal podocytes in childhood-onset active and inactive lupus nephritis

BACKGROUND Childhood-onset systemic lupus erythematosus (SLE) is frequently complicated with lupus nephritis (LN), which is characterized by the deposition of DNA-containing immune complex to the glomerulus. Toll-like receptor 9 (TLR9), capable of recognizing the microbially derived CpG oligonucleotide, plays a crucial role in the innate immunity. TLR9 is also assumed to be related to the aetiology of SLE in the recognition of anti-DNA antibody-containing immune complex, but this remains controversial. We conducted a study to elucidate the association between TLR9 and LN in childhood-onset SLE. METHODS We compared the expression and localization of TLR9 and the slit membrane-related protein in the biopsied kidney sample by immunostaining in four children with active or inactive LN. We also evaluated their laboratory findings, such as anti-DNA antibody, complement and proteinuria at biopsy, to assess the correlation to the findings of the immunostaining. RESULTS TLR9 is not expressed in a normal control kidney. However, TLR9 develops in podocytes only in active LN but disappears in remission. Meanwhile, the slit membrane-related proteins such as nephrin, podocin and synaptopodin in podocytes express clearly and uniformly in remission, but their expression is markedly diminished in active LN, which results in podocyte injury. When TLR9 is expressed in podocytes, all the patients simultaneously showed hypocomplementaemia, high titre of anti-double-stranded DNA (dsDNA) antibody and proteinuria. CONCLUSION Injured podocytes in active LN express TLR9. This expression could be associated with proteinuria and increased anti-dsDNA antibody. This is the first report indicating that TLR9 is involved in the aetiology of LN and that it may play some role in podocyte injury.

Intrarenal suppression of angiotensin II type 1 receptor binding molecule in angiotensin II-infused mice

ATRAP [ANG II type 1 receptor (AT1R)-associated protein] is a molecule which directly interacts with AT1R and inhibits AT1R signaling. The aim of this study was to examine the effects of continuous ANG II infusion on the intrarenal expression and distribution of ATRAP and to determine the role of AT1R signaling in mediating these effects. C57BL/6 male mice were subjected to vehicle or ANG II infusions at doses of 200, 1,000, or 2,500 ng·kg(-1)·min(-1) for 14 days. ANG II infusion caused significant suppression of ATRAP expression in the kidney but did not affect ATRAP expression in the testis or liver. Although only the highest ANG II dose (2,500 ng·kg(-1)·min(-1)) provoked renal pathological responses, such as an increase in the mRNA expression of angiotensinogen and the α-subunit of the epithelial sodium channel, ANG II-induced decreases in ATRAP were observed even at the lowest dose (200 ng·kg(-1)·min(-1)), particularly in the outer medulla of the kidney, based on immunohistochemical staining and Western blot analysis. The decrease in renal ATRAP expression by ANG II infusion was prevented by treatment with the AT1R-specific blocker olmesartan. In addition, the ANG II-mediated decrease in renal ATRAP expression through AT1R signaling occurred without an ANG II-induced decrease in plasma membrane AT1R expression in the kidney. On the other hand, a transgenic model increase in renal ATRAP expression beyond baseline was accompanied by a constitutive reduction of renal plasma membrane AT1R expression and by the promotion of renal AT1R internalization as well as the decreased induction of angiotensinogen gene expression in response to ANG II. These results suggest that the plasma membrane AT1R level in the kidney is modulated by intrarenal ATRAP expression under physiological and pathophysiological conditions in vivo.

Over‐expression of PAR‐3 suppresses contact‐mediated inhibition of cell migration in MDCK cells

Background: PAR‐3 is one of the PAR proteins, previously named ASIP, which are indispensable for the establishment of cell polarity in the embryo as well as differentiated epithelial cells. In mammalian epithelial cells, it forms a ternary complex with aPKC and PAR‐6, and is localized to the tight junction that has been suggested as being important for creating cell polarity.