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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.

ABSTRACT: 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.

SUBMITTER: Higgins PJ

PROVIDER: S-EPMC1135552 | biostudies-other | 1989 Jan

REPOSITORIES: biostudies-other

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Project description:The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71?kPa) and tissue culture plastic (TCP; > 1?GPa) in media containing 0 or 10?ng/ml TGF?1 for 72?h. Cells were treated with 0.4??M Lat-B or DMSO for 30?min every 24?h for 72?h. RNA was collected from cells and expression of alpha-smooth muscle actin (?-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess ?-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n?=?3) or 25% DMSO (vehicle control, n?=?3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less ?SMA mRNA (P???0.007) for RCF cells grown on 22 and 71?kPa substrates as well as TCP without or with TGF?1, and significantly decreased ?-SMA protein expression in RCFs cultured on the intermediate (22?kPa) stiffness in the absence (P?=?0.028) or presence (P?=?0.018) of TGF?1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P?>?0.05) in the absence or presence of TGF?1, but RCFs grown on stiff hydrogels (71?kPa) had significantly more keratocan mRNA expression versus the 22?kPa hydrogel or TCP (P?<?0.001) without TGF?1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of ?-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.

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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.

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