Several proteases and their co-expressed inhibitors modulate the interdependent processes of cell migration and matrix proteolysis during wound repair. Transcription of the gene encoding plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor important in the control of barrier proteolysis and cell-to-matrix adhesion, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. Using a well-defined culture model of acute epidermal wounding and reepithelialization, PAI-1 mRNA/protein synthesis was induced early after monolayer scraping and restricted to cells comprising the motile cohort. PAI-1 levels in locomoting cells remained elevated (relative to the distal,contact-inhibited monolayer regions) throughout the time course of trauma repair. Targeted PAI-1 downregulation by transfection of antisense PAI-1 expression constructs significantly impaired keratinocyte migration and monolayer scrape wound closure. Injury-induced PAI-1 transcription closely paralleled growth state-dependent controls on the PAI-1 gene. An E-box motif(CACGTG) in the PAI-1 proximal promoter (located at nucleotides -160 to -165),previously shown to be necessary for serum-induced PAI-1 expression, was bound by nuclear factors from wound-stimulated but not quiescent, contact-inhibited,keratinocytes. UV crosslinking approaches to identify E-box-binding factors coupled with deoxyoligonucleotide affinity chromatography and gel retardation assays confirmed at least one major E-box-binding protein in both serum- and wound-activated cells to be USF-1, a member of the helix-loop-helix family of transcription factors. An intact hexanucleotide E-box motif was necessary and sufficient for USF-1 binding using nuclear extracts from both serum- and wound-simulated cells. Two species of immunoreactive USF-1 were identified by western blotting of total cellular lysates that corresponded to the previously characterized phosphorylated and non-phosphorylated forms of the protein. USF-1 isolated by PAI-1 promoter-DNA affinity chromatography was almost exclusively phosphorylated. Only a fraction of the total cellular USF-1 in proliferating cultures, by comparison, was phosphorylated at any given time. PAI-1 E-box binding activity, assessed by probe mobility shift criteria,increased within 2 hours of monolayer scrape injury, a time frame consistent with wound-stimulated increases in PAI-1 transcription. Relative to intact cultures, scrape site-juxtaposed cells had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity correlating with the specific in situ-restricted expression of PAI-1 transcripts/protein in the wound-edge cohort. USF-1 immunocytochemical staining declined significantly with increasing distance from the denudation site. These data are the first to indicate that binding of USF-1 to its target motif can be induced by `tissue'injury in vitro and implicate USF-1 as a transcriptional regulator of genes(e.g. PAI-1) involved in wound repair.
Vorinostat, a novel HDAC inhibitor, is efficacious and well tolerated in patients with CTCL and is being investigated for its efficacy and safety in other types of cancers and as a part of combination therapy.
Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the SERPIN gene family that functions to regulate the plasmin-based pericellular proteolytic cascade, is growth state-regulated in normal rat kidney (NRK) cells (Ryan and Higgins, 1990, J. Cell. Physiol., 155:376-384; Ryan et al., 1996, Biochem. J., 314:1041-1046). Comparative analysis of arrest states induced in NRK cells upon exposure to serum-deficient (0.5% FBS) or serum-free culture conditions served to define the kinetics of PAI-1 gene expression and fate of de novo-synthesized PAI-1 protein. While cells rendered quiescent in serum-free or serum-deficient media were equivalent with regard to the time course of PAI-1 mRNA induction, the level of expressed transcripts (27-fold vs. 12-fold) and accumulated saponin fraction PAI-1 protein (12-fold vs. 6-fold) were consistently greater in cells recruited into exponential growth phase from a serum-free as compared to a serum-deficient arrest condition. Relative PAI-1 mRNA abundance increased within 1-2 hr post-serum addition, was maximal at 4 hr, and declined rapidly thereafter; this time course of expression coupled with placement of entry into DNA synthetic phase at approximately 12 hr after stimulation indicates that PAI-1 induction is an early-to-mid G1 phase event. Induced PAI-1 protein was evident immunocytochemically within 2 hr of serum stimulation as a peripheral "rim" of accumulated protein restricted to the cellular ventral surface at the plane of the substrate. No PAI-1 was detected between individual cells suggesting that this protein may be targeted directly to the undersurface region. By 6 hr post-stimulation, the rim of PAI-1 deposition increased in intensity and broadened to occupy approximately 30 to 50% of the total undersurface area. Double-label immunocytochemistry indicated that accumulated PAI-1 was deposited in close proximity to, but not actually within, vinculin-containing focal contact structures. Potential functionality of induced PAI-1 expression to either the initiation or maintenance of the serum-stimulated phenotype was assessed using antibodies to PAI-1. The IgG fractions of two different antisera which neutralize the ability of PAI-1 to complex with and thereby inhibit the catalytic activity of urokinase plasminogen activator significantly reduced (by 25-35%) the incidence of cells displaying the serum-stimulated phenotype; antibodies that bind PAI-1 but do not block PAI-1 inhibitory activity were without effect. In view of the vagaries of antibody accessibility and in situ neutralizing activity (particularly in a region as structurally complex as the focal contact), these data may actually underestimate the importance of PAI-1 in maintaining the activated phenotype.
Antibody production tests have traditionally been used to test biological materials for viral contamination. Now molecular biology techniques have emerged as an alternative. The authors compare MAP testing with PCR-based detection methods, focusing on differences in animal use, laboratory requirements, sample size, and limits of detection.
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