Stephen P. Sugrue

A Novel Repressor, par-4, Modulates Transcription and Growth Suppression Functions of the Wilms' Tumor Suppressor WT1

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Histopathologic Features of the Floppy Eyelid Syndrome: Involvement of Tarsal Elastin

Purpose: Patients with the floppy eyelid syndrome have chronic papillary conjunctivitis with easily everted upper eyelids and a soft, pliant upper tarsus. The purpose of this study is to describe the clinical features and the histopathologic correlate in a group of patients with floppy eyelid syndrome. Methods: The authors examined eight patients with floppy eyelid syndrome, four of whom underwent surgical management with horizontal eyelid shortening. Eyelid tissue from these patients was examined using light microscopy, electron microscopy, and immunohistochemistry and compared with controls with unrelated eyelid or orbital disorders. Results: Clinical findings included obesity or eye rubbing, lash ptosis, and, less commonly, blepharoptosis. Two patients had documented sleep apnea with abnormal sleep electroencephalogram. Light microscopy of the surgical specimens showed chronic conjunctival inflammation, papillary conjunctivitis, and meibomian gland abnormalities, including granuloma formation. Verhoeffs modified elastin stain demonstrated a marked decrease in the amount of elastin fibers in tarsus from patients with floppy eyelid syndrome compared with controls. Immunohistochemical staining for elastin also showed a marked decrease of tarsal elastin in floppy eyelid patients compared with controls. In contrast, immunohistochemical stains showed that the distribution of collagen types I and III was similar between patients with floppy eyelid syndrome and controls. Electron microscopy demonstrated that tarsal collagen was comparable in patients and controls, and that there was a reduced amount of tarsal elastin in floppy eyelid syndrome compared with controls. Conclusions: These findings demonstrate that tarsal elastin is decreased in the floppy eyelid syndrome, which may contribute to the laxity of the tarsus in this disorder.

Interaction of embryonic corneal epithelium with exogenous collagen, laminin, and fibronectin: role of endogenous protein synthesis.

Abstract Previously, we have shown that the embryonic corneal epithelium is capable of interacting with exogenous collagen, laminin, and fibronectin in soluble form, each of which causes isolated epithelium cultured on Millipore filter to stop blebbing, reorganize the basal cytoskeleton, and flatten. Here we examine the involvement of endogenously derived extracellular matrix (ECM) molecules in the interaction of the basal epithelial cell surface with the added ECM molecules. We demonstrate here that the isolated avian corneal epithelium cultured on Millipore filter is capable of synthesizing collagens and laminin, but not fibronectin. To examine whether the epithelium is capable of interacting directly with exogenous ECM components or if there is the necessity for production of a linker molecule, epithelial protein synthesis was inhibited with cycloheximide (CHX). The blebbing epithelium in the presence of CHX was then confronted with soluble ECM molecules added to the medium under the filter; such epithelia are able to interact with, and flatten in response to, both collagen and laminin. However, such inhibited epithelia continue to bled in the presence of fibronectin. We next used l -azetidine-4-carboxylic acid (LACA) to interfere with collagen secretion. Epithelia exposed to LACA are still capable of interacting with collagen and laminin, but not fibronectin, indicating a dependence on collagen secretion. These results suggest that fibronectin requires a linker protein, probably collagen, to interact with the basal epithelial surface, whereas both collagen and laminin may interact directly with the cell surface to transform the basal cytoskeleton into the cortical mat typical of differentiating corneal epithelium in situ.

Temporal and spatial distribution of type XII collagen in high cell density culture of periosteal-derived cells

Periosteal-derived cells of young chicks have been reported to possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes under high cell density culture conditions. In this culture, the temporal and spatial distribution of type XII collagen was immunocytochemically assessed using a monoclonal antibody. These high-density plated cells first formed a multilayer of fibroblast-like cells, in which type I and XII collagen were evenly distributed throughout the full thickness of the culture. With time, the top portion of the culture differentiated into bone tissue, while cells below this top layer differentiated into hypertrophic chondrocytes. In this transition, type XII collagen was temporally and spatially colocalized primarily with type I collagen: the top portion of bone layer was positive for both type I and XII collagens, whereas their staining intensity in the bottom portion decreased with time in culture. Using this antibody, type XII collagen was also found in developing embryonic chick tibiotarsus. These observations, taken together, suggest that type XII collagen production is a characteristic property of bone-forming cells.

Corepressor CtBP and Nuclear Speckle Protein Pnn/DRS Differentially Modulate Transcription and Splicing of the E-Cadherin Gene

ABSTRACT CtBP is a transcriptional corepressor with tumorigenic potential that targets the promoter of the tumor suppressor gene E-cadherin. Pnn/DRS (Pnn) is a “nuclear speckle”-associated protein involved in mRNA processing as well as transcriptional regulation of E-cadherin via its binding to CtBP. Here, we show that CtBP can recruit Pnn to CtBP-associated complexes, resulting in Pnn-dependent chromatin remodeling at the E-cadherin promoter. In addition, CtBP and Pnn can differentially modulate E-cadherin mRNA splicing, with polymerase II serving as an interface in this event. Therefore, the Pnn/CtBP functional interplay represents a novel mechanism linking the corepressor CtBP and Pnn to the transcription-coupled mRNA splicing of a major tumor suppressor gene. Our findings implicate the existence of the molecular switches involved in tumorigenesis, which coordinate promoter-specific events and mRNA processing, by serving as bridging elements between the regulatory complexes both at gene promoters and within the mRNA splicing machineries.