The effect of mechanical strain on protease production by keratinocytes.

BACKGROUND: Intact skin is under constant tension, transmitted from the underlying dermis, but when tension is lost (i.e. upon wounding) protease activity is upregulated. OBJECTIVES: To investigate the effect of mechanical strain on protease production by both normal and transformed keratinocytes in vitro. METHODS: Keratinocytes were seeded on to membranes precoated with either type I or type IV collagen. After 48 h medium was replaced with serum-free medium and mechanical strain was applied. RESULTS: Mechanical strain resulted in decreased urokinase-type plasminogen activator (uPA) production by normal human keratinocytes (P<0.05) but increased production by transformed keratinocytes (P<0.05) cultured on type I and type IV collagen. CONCLUSIONS: Differential production of uPA by normal and transformed keratinocytes is relevant in the context of normal function, wound healing and tumorigenesis.

The Y-box binding protein YB-1 suppresses collagen alpha 1(I) gene transcription via an evolutionarily conserved regulatory element in the proximal promoter.

Appropriate expression of collagen type I, a major component of connective tissue matrices, is dependent on tight transcriptional control and a number of trans-activating and repressing factors have been characterized. Here we identify the Y-box binding protein-1 (YB-1) as a novel repressor of the collagen type alpha1(I) (COL1A1) gene. Collagen type I mRNA and protein levels decreased upon overexpression of YB-1 by transfection in NRK fibroblasts. The human, rat, and mouse COL1A1 promoter -220/+115 contains three putative Y-boxes, one of these sites, designated collagen Y-box element (CYE), includes a Y-box plus an adjacent 3' inverted repeat. DNase-I footprinting and Southwestern blotting with fibroblast nuclear extract demonstrated binding of several nuclear proteins across the CYE, one of which was identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel shift assays with a preference for single-stranded templates. The entire sequence (-88/-48) was required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by supershift studies. COL1A1 promoter-reporter constructs were suppressed up to 80% by cotransfection with YB-1 in a variety of cell types. In addition, CYE conferred YB-1 responsiveness on two heterologous promoters further demonstrating the importance of this repressor region. Mung bean nuclease sensitivity analysis suggested that repression is most likely exerted through changes in DNA conformation.

The recurring evolution of lipoprotein(a). Insights from cloning of hedgehog apolipoprotein(a).

The lipoprotein Lp(a), a major inherited risk factor for atherosclerosis, consists of a low density lipoprotein-like particle containing apolipoprotein B-100 plus the distinguishing component apolipoprotein(a) (apo(a)). Human apo(a) contains highly repeated domains related to plasminogen kringle four plus single kringle five and protease-like domains. Apo(a) is virtually confined to primates, and the gene may have arisen during primate evolution. One exception is the occurrence of an Lp(a)-like particle in the hedgehog. Cloning of the hedgehog apo(a)-like gene shows that it is distinctive in form and evolutionary history from human apo(a), but that it has acquired several common features. It appears that the primate and hedgehog apo(a) genes evolved independently by duplication and modification of different domains of the plasminogen gene, providing a novel type of "convergent" molecular evolution.

C/T polymorphism in the 5' untranslated region of the apolipoprotein(a) gene introduces an upstream ATG and reduces in vitro translation.

Elevated plasma levels of lipoprotein(a) [Lp(a)] are a significant-independent risk factor for arteriosclerosis. Interindividual levels of Lp(a) vary nearly 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. A search was made for sequence variants in the 5' flanking region of the apo(a) gene that affect its expression. A C to T transition at position +93 from the transcription start site was found with a frequency of 14% in the study population. In transient transfection assays in HepG2 cells, luciferase reporter gene constructs with a T at this position were associated with a 58% reduction in luciferase activity compared with the more common allele. This single base variant had no significant effect on the binding of nuclear regulatory proteins; however, it introduced an additional upstream ATG initiation codon with its own in-frame stop codon. Furthermore, equivalent levels of mRNA were produced in HepG2 cells transfected with reporter gene constructs containing either a T or a C at position +93. In vitro translation experiments using transcripts derived from either variant apo(a) promoter revealed a 60% reduction in translation associated with the T allele. Hence, the additional ATG created by the T at position +93 in the 5' flanking region of the apo(a) gene impairs the efficiency of translation from the bona fide ATG initiation codon.

Apolipoprotein(a) gene transcription is regulated by liver-enriched trans-acting factor hepatocyte nuclear factor 1 alpha.

Elevated levels of lipoprotein(a) (Lp(a)) in the plasma are a risk factor for coronary artery disease and stroke. Plasma Lp(a) concentrations are highly heritable and predominantly determined by the liver-specific apolipoprotein(a) (apo(a)) gene. In this report we show by deletion analysis that sequences from -98 to +130 of the apo(a) gene are sufficient to direct liver-specific transcription. DNase I protection analysis of this region using HepG2 nuclear extracts revealed six major protein-binding sites, designated A to F. A mutation within footprint C, situated in the 5'-untranslated region of the gene, resulted in a marked reduction of luciferase expression from a reporter construct to 12% of wild type. This was not due to a decrease in mRNA stability. Gel mobility shift assays demonstrated that site C binds hepatocyte nuclear factor 1 alpha (HNF-1 alpha), and overexpression of HNF-1 alpha in HepG2 cells resulted in a significant stimulation of transcription from this promoter fragment. Mutation of footprint B resulted in a 2-fold enhancement of transcription. These results show that positive regulation of transcription of the apo(a) gene is dependent on the binding of HNF-1 alpha to a regulatory element situated downstream of the mRNA start site, and suggest that an as yet unidentified protein may negatively regulate apo(a) transcription by binding to a discrete sequence within the 5'-untranslated region.

5' control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family.

Elevated blood levels of apolipoprotein(a), the component of lipoprotein(a) that distinguishes it from low density lipoprotein, are a major risk factor for atherosclerosis. The apolipoprotein(a) gene is highly similar to the plasminogen gene and to at least four other genes or pseudogenes. The 5' untranslated and flanking sequences of these six genes contain extensive regions of near identity and share sequence elements involved in the initiation of transcription and translation. About 1000 base pairs of flanking DNA of each gene are sufficient to promote transcription in cultured hepatocytes. The apolipoprotein(a) gene promoter contains functional interleukin 6-responsive elements, consistent with the reported acute-phase response of apolipoprotein(a). Flanking genomic fragments of the apoliprotein(a) gene from two individuals with vastly different plasma apolipoprotein(a) concentrations have sequence differences that are reflected in differences in the rate of in vitro transcription.