Tài liệu Báo cáo khoa học: Oxygen tension regulates the expression of a group of procollagen

Oxygen tension regulates the expression of a group of procollagen

hydroxylases

Karl-Heinz Hofbauer1

, Bernhard Gess1

, Christiane Lohaus2

, Helmut E. Meyer2

, Do¨ rte Katschinski3

and Armin Kurtz1

1

Institut fu¨r Physiologie der Universita¨t Regensburg, Germany; 2

Medizinisches Proteom, Center der Ruhr, Universita¨t Bochum,

Germany; 3

Abteilung Zellphysiologie der Martin-Luther Universita¨t Halle, Germany

In this study, we have characterized the influence of hypoxia

on the expression of hydroxylases crucially involved in col￾lagen fiber formation, such as prolyl-4-hydroxylases (Ph4)

and procollagen lysyl-hydroxylases (PLOD). Using the rat

vascular smooth muscle cell line A7r5, we found that an

hypoxic atmosphere caused a characteristic time-dependent

five- to 12-fold up-regulation of the mRNAs of the two P4h

a-subunits [aI (P4ha1) and aII (P4ha2)] and of two lysyl￾hydroxylases (PLOD1 and PLOD2). These effects of hyp￾oxia were mimicked by the iron-chelator deferoxamine

(100 lM) and by cobaltous chloride (100 lM). The hypoxic

induction of these genes was also seen in the mouse juxta￾glomerular As4.1 cell line and mouse hepatoma cell line

Hepa1 but was almost absent in the mutant cell line

Hepa1C4, which is defective for the hypoxia-inducible

transcription factor 1 (HIF-1). In addition, the enzyme

expression was induced by hypoxia in mouse embryonic

fibroblasts but not in embryonic fibroblasts lacking the HIF￾1a subunit. These findings indicate that hypoxia stimulates

the gene expression of a cluster of hydroxylases that are

indispensible for collagen fiber formation. Strong indirect

evidence, moreover, suggests that the expression of these

enzymes during hypoxia is coordinated by HIF-1.

Keywords: prolyl hydroxylase; lysyl hydroxylase; protein

disulfide isomerase; hypoxia inducible transcription factor.

In a variety of tissues, an hypoxic environment favors the

formation of collagen deposits. Such an hypoxia-related

collagen formation has a clear (patho)physiological impact

for wound healing in the skin, for the remodeling of small

muscular pulmonary arteries in hypoxia-induced pulmonary

hypertension and possibly also for cardiac hypoxia. The

formation of collagen fibers and deposits is a multi-step

event that includes procollagen protein synthesis, prolyl

hydroxylation as requirement for triple helix formation, lysyl

hydroxylation, protein folding, maturation and secretion,

and finally covalent cross-bridging between collagen fibers

through the activity of the lysyloxidase. Which of these steps

are directly triggered by hypoxia and how this is accom￾plished is not well understood. It has been reported that

hypoxia increases mRNA levels for different procollagens in

the lung [1,2] and heartin vivo [3].In vitro studies suggest that

this effect of hypoxia on procollagen gene expression might

be isoform and cell-type specific. Thus, hypoxia stimulates

procollagen I formation in renal [4], dermal [5], and cardiac

fibroblasts [6], but neither in fetal lung fibroblasts [7] nor in

3T3 fibroblasts [8]. 3T3 fibroblasts [8], like renal mesangial

cells [9], however, increase the gene expression of procol￾lagen IV in response to hypoxia. The effect of hypoxia on the

activity of the prolyl-4-hydroxylase (PHD-4 or P4h) is

clearer; it is crucially required to enable triple helix formation

and has been found to be increased in its activity in response

to hypoxia [7,10–12]. For the PHD-4(P4h) heterotetramer

enzyme (a2b2) there exist two isoforms with a variable

a-subunit (aI or aII) and a constant b-subunit, which is

identical to protein disulfide-isomerase (PDI) [13]. In vitro

studies have shown recently a moderate increase of aI

protein and gene expression in fetal lung fibroblasts during

hypoxia [14], which is likely mediated by the hypoxia

inducible transcription factor HIF-1 [14]. Whether hypoxia

also triggers the gene expression of aII is not yet known.

Although PDI as the b-subunit is considered to be expressed

in excess, there is a report that hypoxia also causes a delayed

increase of PDI expression in cultured astrocytes [15].

Whether such an hypoxic stimulation of PDI expression is a

more general phenomenon and what the possible underlying

mechanism could be, is also unknown. In addition to prolyl

hydroxylation, maturation of procollagen also requires the

hydroxylation of lysin residues mediated by procollagen

lysyl-hydroxylases (PLOD), for which three isoforms exist

[16], two of which, namely PLOD1 and PLOD2, are more

closely related and colocalize with P4h in the endoplasmic

reticulum [17]. Whether the homodimeric PLODs are

triggered by hypoxia is also unknown.

Screening a rat vascular smooth muscle cell line for

hypoxia-induced proteins revealed a clear stimulation of

P4ha1 and P4ha2 protein expression that was absent in a cell

line defective for HIF-1. As these findings suggested a more

Correspondence to A. Kurtz, Institut fu¨r Physiologie, Universita¨t

Regensburg, D-93040 Regensburg, Germany.

Fax: + 49 941 9434315, Tel.: + 49 941 9432980,

E-mail: [email protected]

Abbreviations: HIF-1, hypoxia inducible transcription factor 1; PDI,

protein disulfide isomerase; Ph4, prolyl-4-hydroxylases; PLOD, pro￾collagen lysyl-hydroxylases; SDS/PAGE, sodium dodecyl sulfate/

polyacrylamide gel electrophoresis; UPR, unfolded protein response.

(Received 31 July 2003, revised 8 September 2003,

accepted 19 September 2003)

Eur. J. Biochem. 270, 4515–4522 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03846.x