Cytochalasin D-mediated hyperinduction of the substrate-associated 52-kilodalton protein p52 in rat kidney fibroblasts

Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.     

Growth state-regulated expression of p52(PAI-1) in normal rat kidney cells

In normal rat kidney (NRK) cells, synthesis of the 52-kDa substrate-associated type 1 inhibitor of plasminogen activator [p52(PAI-1)] is linked to alterations in cell shape and substrate adhesion. Subconfluent NRK cells accumulated significantly more ventral undersurface-associated p52(PAI-1) compared to newly confluent or 1 - to 2-day postconfluent cultures, suggesting that p52(PAI-1) expression was also growth state-modulated. Since cytoarchitectural constraints function in cell growth control, changes in p52(PAI-1) synthesis were assessed with respect to defined morphologic events that accompany growth activation of cultured NRK cells. Stimulation of low population density, quiescent NRK cells with 20% serum-containing medium resulted in a rapid increase in matrix p52(PAI-1) protein content (6- and 26-fold after 1 and 5 hr, respectively). Growth activation in response to serum reflected elevations in p52(PAI-1) cytoplasmic mRNA abundance, which peaked at 2 hr (125-fold increase) and subsequently declined (100-fold increase) at 5 hr poststimulation. Morphologic analysis indicated that quiescent NRK cells were devoid of transcytoplasmic actin filaments and focal contact-associated vinculin. A marked increase in the fraction of cells that eleborated transcytoplasmic microfilaments and vinculin-containing focal adhesions was evident within 5 min of serum addition. Such cytoarchitectural restructuring preceded p52(PAI-1) induction. Morphologic reorganization and p52(PAI-1) induction occurred prior to progression of cells through the S-phase, indicating they are early events associated with serum stimulation in the NRK cell system. The relevance of p52(PAI-1) induction during this growth state transition is not clear but may influence the established cytoarchitectural changes observed prior to p52(PAI-1) induction by regulating pericellular proteolysis and, thereby, cell-to-substrate adhesion. © 1993 Wiley-Liss, Inc.     

Sodium-n-butyrate induces secretion and substrate accumulation of p52 in Kirsten sarcoma virus-transformed rat kidney fibroblasts

1. A group of five transformation-responsive secreted proteins, ranging in molecular mass from 31 to 70 kDa, were identified in cultured normal rat kidney (NRK) fibroblasts. 2. One such protein (p52) was found to be a major secreted and substrate-attached component of NRK cells. 3. Kirsten sarcoma virus-transformed NRK cells failed to accumulate p52 in either the secreted or substrate-associated protein compartments; this protein was inducible, however, in transformed cells by culture in 2 mM sodium-n-butyrate. 4. Kinetics of p52 induction in transformed NRK cells, relative to the time course of increased cell spreading, and its enrichment in the substrate-associated protein fraction suggest that p52 might function in cell-substrate attachment.     

Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts. Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells.

A 52 kDa protein (p52) was identified, using differential extraction and electrophoretic criteria, as a major extracellular and substrate-associated component of normal rat kidney (NRK) fibroblasts. Cells transformed with Kirsten murine sarcoma virus (KNRK cells) did not express p52 constitutively, but were inducible for both p52 production and its substrate association during culture in sodium butyrate (NaB)-supplemented growth medium. Comparative analysis of the relative molecular mass, subcellular distribution, and isoelectric complexity (five variants ranging in pI from 5.4 to 6.2) of the 52 kDa species constitutively and inducibly expressed by NRK and KNRK/NaB cells respectively, indicated that they were, indeed, the same protein. p52 selectively localized to cellular fractions enriched in substrate focal contact sites and associated ventral undersurface components. NaB induction of p52 in KNRK cells occurred before cell spreading; other polar compounds, such as dimethyl sulphoxide, which did not induce KNRK cell spreading, similarly failed to elicit p52 production. p52 accumulated more rapidly in (and was quickly released from) the focal-contact-enriched protein fraction of NRK cells compared with its time course of appearance in the medium. These data collectively suggest that p52 is one of a relatively small number of proteins the synthesis of which is either involved in determination of cell shape or regulated as a consequence of cell-shape changes.     

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.     

Expression of macrophage p120 depends on early protein synthesis

We have previously identified a group of early proteins preceding the expression of a 120-kDa protein (p120) which coincides with tumoricidal activation in peritoneal macrophages. In the present report, we have asked whether the in vitro induction of new or enhanced expression of p120 depends on early protein synthesis and RNA synthesis during the treatment period. Expression of p120 was sensitive to pretreatment of the macrophages with either actinomycin D or cycloheximide, indicating that both active protein synthesis and RNA synthesis were required. When poly-adenylated RNA isolated from various macrophage populations was translated in a rabbit reticulocyte in vitro translation system, only mRNA isolated from cells which express p120 was able to direct synthesis of a 120-kDa polypeptide. This product showed identical mobility to p120 induced in intact activated macrophages radiolabeled with [35S]methionine. The presence of translatable p120 mRNA was dependent upon treatment of thioglycollate-elicited macrophages with both IFN-gamma plus LPS at low doses, as is expression of p120 in intact cells. Accumulation of translatable p120 mRNA was blocked by treatment with cycloheximide, indicating that active protein synthesis was required during the induction period. These results suggest that the presence of specific translatable mRNA encoding the p120 polypeptide is dependent upon the expression of early macrophage gene products.     

Role of p52 (NF-kappaB2) in LPS tolerance in a human B cell line

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.     

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.     

The substrate-associated protein p45 of porcine endothelial cells: multiple isoforms, cytoskeletal-like properties and induction by hyperoxic stress

1. Cultured mesenchymal cells respond to hyperoxic (hyper-O2) stress with increased cell flattening/substrate adhesion and overall 47-69% reductions in total matrix-associated (i.e. saponin-resistant [SAP fraction]) protein. 2. Electrophoretic analysis revealed a selective hyper-O2-related 2.7- to 4-fold increase in SAP and cytoskeletal fraction deposition of the protein p45 beginning early (within 12 hr) after initial exposure of porcine endothelial cells to hyper-O2 and increasing over a 48 hr period. 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-O2-augmented matrix deposition of 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-02-augmented matrix deposition of p45 involved all 8 isoforms with the more basic subtypes exhibiting slightly greater net increases. 4. Both the specificity and time course of p45 induction, relative to the onset of hyper-O2 cytoarchitectural remodeling, indicate that p45 up-regulation constitutes an early aspect of the hyper-O2 adaptive response.