Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: Implications for collagen cross-linking and treatment of fibrosis
Introduction
The synthesis of collagen is a multi-step process, involving several unique post-translational modifications essential for the stabilisation of the collagen molecule. The assembly of collagen molecules into the functional unit, the collagen fibril, is further stabilised by covalent intermolecular cross-links. Hydroxylation of lysine residues is one of the key steps in the maturation of collagen, providing specific sites for glycosylation (Kadler, 1994) and determining the chemical nature of the intermolecular cross-links (Eyre, 1987). A family of lysyl hydroxylases (LH; EC 1.14.11.4), consisting of LH1, LH2a, LH2b, and LH3, is responsible for the catalytic conversion of lysine into hydroxylysine (Hautala et al., 1992a, Pasquali et al., 1997, Passoja et al., 1998, Risteli et al., 2004, Valtavaara et al., 1997, Valtavaara et al., 1998, Yeowell and Walker, 1999). Mutations in LH1 lead to the clinical phenotype of Ehlers–Danlos syndrome type VI (EDS VI; OMIM#225400) (Yeowell and Walker, 2000), which is biochemically characterised by a deficiency in triple helical hydroxylysine residues (Steinmann et al., 1995). An almost complete lack of hydroxylated lysine residues in the collagen telopeptides is seen in Bruck syndrome (BS; OMIM#259450) bone (Bank et al., 1999) and in a number of BS patients LH2 is mutated (Ha-Vinh et al., 2004, van der Slot et al., 2003). These connective tissue disorders indicate substrate specificity for the different lysyl hydroxylases and their clinical phenotypes show the devastating effect of disturbed hydroxylation of triple helical or telopeptide lysine residues on tissue performance. The specificity of LH1 is likely directed towards the lysine residues in the helical domain of the collagen molecule (Pasquali et al., 1997). There is a growing body of evidence that LH2b is involved in the hydroxylation of telopeptide lysine residues (Mercer et al., 2003, Pornprasertsuk et al., 2004, Uzawa et al., 1999, van der Slot et al., 2003, van der Slot et al., 2004). No mutations in LH3 have been identified so far that can be linked to a disorder of the extracellular matrix and its lysyl substrate specificity is as yet unknown. LH3 is different from the other two lysyl hydroxylases in that it also displays glucosyl and galactosyl transferase activity, thereby contributing to the glycosylation of hydroxylysine residues in the collagen molecule (Heikkinen et al., 2000, Wang et al., 2002). The embryonic lethality of LH3 knock-out mice shows that this lysyl hydroxylase provides essential functions in the body (Rautavuoma et al., 2004).
The hydroxylation status of the cross-linking residue in the collagen telopeptide, being either lysine or hydroxylysine, determines the route of intermolecular enzymatic cross-linking. In the allysine cross-linking route lysine is converted by lysyl oxidase (LOX) into the aldehyde allysine, whereas in the hydroxyallysine route hydroxylysine is converted into the aldehyde hydroxyallysine. These aldehydes further react with a lysine, hydroxylysine, or histidine in the triple helix to form di-, tri-, or tetra-functional cross-links (Eyre et al., 1984b, Reiser et al., 1992, Robins, 1982). The mature cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are formed via the hydroxyallysine route and differ only with respect to the triple helical residue involved in the cross-link, hydroxylysine and lysine, respectively (Eyre, 1987). These kinds of cross-links occur in a variety of connective tissues such as bone, cartilage, tendon, and ligaments. In contrast, skin collagen contains predominantly allysine-derived cross-links and only minor amounts of hydroxyallysine-derived cross-links (Eyre et al., 1984a). In general, the prevalence of a certain type of cross-link appears to be tissue-specific and is involved in providing tissues with certain properties (Banse et al., 2002, Wassen et al., 2000).
In fibrotic disorders, which are characterised by an accumulation of collagen, an increase in hydroxyallysine cross-linking is observed (Bailey et al., 1975, Brenner et al., 2000, Istok et al., 2001, Last et al., 1990, Ricard-Blum et al., 1992). There are strong indications that these cross-links diminish the susceptibility of collagen to proteolytic degradation and in this way contribute to the unwanted collagen accumulation in fibrosis (Bailey et al., 1975, Bailey and Light, 1985, Last et al., 1990, Ricard-Blum et al., 1992, Ricard-Blum et al., 1993). It is therefore plausible that redirecting collagen cross-linking towards the allysine route, by reducing the overhydroxylation of telopeptide lysine residues, prevents the irreversible deposition of collagen. We have previously shown that an elevated LH2b expression in fibrotic cells is associated with an increase in pyridinoline cross-links in the collagen deposited by these cells. These data indicate that induction of LH2b expression leads to overhydroxylation of telopeptide lysine residues and the concomitant formation of hydroxyallysine cross-links (van der Slot et al., 2003, van der Slot et al., 2004). Consequently, LH2b becomes a prime target for an anti-fibrosis therapy based on decreasing hydroxyallysine cross-linking.
Minoxidil, an antihypertensive drug, has been reported to reduce lysyl hydroxylase activity by decreasing the LH1 mRNA level (Hautala et al., 1992b, Murad and Pinnell, 1987, Pinnell and Murad, 1987). At the time these studies were performed it was not known that different isoforms of lysyl hydroxylase existed, but it was already postulated that minoxidil would have anti-fibrotic properties by limiting the supply of hydroxylysines for hydroxyallysine cross-link formation (Murad et al., 1994). In the present study we investigated the effect of minoxidil on the gene expression of each individual lysyl hydroxylase, in particular LH2b, and on the cross-linking of collagen to examine whether minoxidil indeed has the potential to decrease hydroxyallysine cross-link formation.
Section snippets
Inhibiting properties of minoxidil on LH gene expression
To unravel the effect of minoxidil on gene expression of the different lysyl hydroxylases, dose- and time-dependent responses were examined in fibroblasts derived from subcutaneous fat of one donor. These fibroblasts were used because of their elevated levels of LH2b gene expression and hydroxyallysine cross-links compared to fibroblasts isolated from dermis (Van Den Bogaerdt, 2004). Concentration-dependent effects on the mRNA levels of LH1, LH2b, and LH3 after a 24 h incubation period were
Discussion
Collagen deposition in human fibrotic tissues often displays an increase in hydroxyallysine-derived cross-links (van der Slot et al., 2004), which most likely contributes to the unwanted accumulation of collagen by conferring increased resistance to proteolytic degradation (Bailey et al., 1975, Bailey and Light, 1985, Last et al., 1990, Ricard-Blum et al., 1992, Ricard-Blum et al., 1993). After the discovery of the inhibitory action on lysyl hydroxylase gene expression, minoxidil was postulated
Fibroblast cultures
Primary fibroblasts obtained from healthy dermis, subcutaneous fat, and keloid were isolated as described in Van Den Bogaerdt et al., 2002. Primary fibroblasts of one keloid donor were obtained from the American Type Culture Collection (ATCC; CRL-1762). Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM; BioWhittaker) containing ultraglutamine I and supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in 5% CO2. Upon
Acknowledgements
We would like to thank Dr. E. Middelkoop from the Association of Dutch Burn Centers (Beverwijk, The Netherlands) for providing split skin- and subcutaneous fat-derived fibroblasts. This study was supported by the Dutch Arthritis Association Grant 99-2-201, the Netherlands Organization for Scientific Research (NWO) Grant 902-27-222, and the Dutch Burns Foundation Grant WO/P04.101.
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