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

Oxygen tension regulates the expression of a group of procollagen
hydroxylases
Karl-Heinz Hofbauer
1
, Bernhard Gess
1
, Christiane Lohaus
2
, Helmut E. Meyer
2
,Do¨ rte Katschinski
3
and Armin Kurtz
1
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 l
M
) and by cobaltous chloride (100 l
M
). 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 heart in 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 (a
2
b
2
) there exist two isoforms with a variable
a-subunit (aIoraII) 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:
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) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03846.x
general HIF-1-related effect of hypoxia on the expression of
the critical hydroxylases for collagen fiber formation, this
study aimed to characterize the influence of hypoxia on the
gene expression of these hydroxylases in more detail.
Materials and methods
Cell cultures
Rat aortic vascular smooth muscle cells (A7r5) from BDXI
rats (ATCC CRL 1444) were cultured in 75 cm
2
flasks
(Sarstedt) with 15 mL Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum and penicillin/strepto-
mycin (P/S; 10 U/10 lgÆmL
)1
)(Biochrom), kept in an
atmosphere of 10% CO
2
(v/v), 21% O
2
and 69% N
2
at

37 °C. Medium was changed every second day and cells were
confluent on days 7–10 after splitting, which was achieved
with trypsin–EDTA for 5 min at 37 °C. For the experi-
ments, cell cultures (triplicates) were incubated in 21% O
2
(i.e. normoxia) or 1% O
2
, 10% CO
2
, 89% N
2
(i.e. hypoxia)
for up to 24 h. Additional culture dishes were incubated at
21% O
2
with either cobalt(II) chloride (100 lmolÆL
)1
)or
with desferoxamine (200 lmolÆL
)1
) for 12 h.
Mouse As4.1 cells [18] were incubated under the afore-
mentioned conditions for 4.5 h.
Mouse hepatoma Hepa1 cells, and their subclone
Hepa1C4, which produces defective ARNT (HIF-1b)[19]
due to a point mutation [20] rendering the cells unable to
form active HIF [21], were grown under the above
mentioned conditions. For the experiments the cells were
incubated either at 0.5% O
2

,10%CO
2
balance N
2
(i.e.
hypoxia) or at 21% O
2
, 10% CO
2
balance N
2
with
deferoxamine (200 lmolÆL
)1
) for 24 h.
Mouse embryonic fibroblasts with normal (+/+) and
with a disrupted (–/–) gene for HIF-1a [22] were grown
under the above-mentioned conditions. The cells were
incubated either at 0.5% O
2
(i.e. hypoxia) or at 21% O
2
with
deferoxamine (100 lmolÆL
)1
) for 24 h.
Preparation of protein samples
After removal of cell culture medium, cell were washed three
times with ice-cold NaCl/P
i

andthenscrapedoffinlysis
buffer (300 lL per 75 cm
2
flask) consisting of 7 molÆL
)1
urea, 2 molÆL
)1
thiourea, 2% CHAPS, 1% dithiothreitol,
Pharmalyte
TM
pH 3–10 (Pharmacia, Uppsala, Sweden),
supplemented with protease inhibitors (CompleteÒ, Boeh-
ringer Mannheim, Germany). The material was then homo-
genized with an Ultraturrax (3 · 10 s) and further sonicated
for 3 · 10 s. The homogenate was then allowed to stand at
room temperature for 60 min prior to ultracentrifugation at
80 000 g at 15 °C for 1 h. Aliquots of the clear supernatant
were frozen in liquid nitrogen and stored at )80 °C. For
determination of the protein concentration, protein was
precipitated with 10% trichloroacetic acid in acetone and
resuspended in 0.1
M
NaOH. Protein concentration was then
determined with the Bio-Rad protein assay (Bio-Rad, Int.).
Two-dimensional PAGE
Protein (150 lg) for silverstained gels and for Coomassie-
blue staining (600 lg) were loaded for each sample onto the
first dimension strips. A linear immobilized pH gradient
(pH 5.0–6.0 IPG buffer 18; Pharmacia) was used as the first
dimension. Hydration of gel strips and sample application

was performed at 50 V for 15 h. For protein separation, a
step-voltage protocol was applied (1 h 150 V, 3 h 500 V,
1 h 1000 V, gradient to 8000 V within 0.5 h). A total volt–
hour product of 60 kVh was used for 150 lgprotein
and 110 kVh for 600 lg protein. Afterwards, the strips
were incubated in 50 mmolÆL
)1
Tris/HCl (pH 6.8), urea
6molÆL
)1
, glycerol 30%, dithiothreitol 65 mmolÆL
)1
,2%
sodium dodecyl sulfate (SDS) for 20 min at room tempera-
ture followed by incubation in 50 mmolÆL
)1
Tris/HCl
(pH 8.8), urea 6 molÆL
)1
, glycerol 30%, iodoacteamide
140 mmolÆL
)1
, and 2% SDS for another 20 min. For the
second dimension, a vertical gradient slab gel of 8–18%
acrylamide was used and SDS/PAGE was performed at
8mApergelat13°C for 4 h followed by 30 mA for 12 h.
At the end of the second dimension, the gels were removed
from the glass plates.
Staining of two-dimensional PAGE
The gels were fixed and stained with silver according to

standard protocols [23]. The gels were then scanned (Image
Scanner Sharp JX-330, Amersham Biosciences) and ana-
lyzed with the
IMAGE
3.1 analysis software package (Amer-
sham Bioscience). Each spot was matched from one gel to
another and the relative volume of matched spots was
compared. For preparative protein analysis higher amounts
of protein were loaded for two-dimensional PAGE and the
protein spots were then stained with colloidal Coomassie-
blue.
Protein sequence analysis
Coomassie-blue stained spots were excised from the gels and
were subjected to ESI-MS analysis [24]. Sequences obtained
with ESI-MS analysis were then compared with the mouse
and rat subset of the NCBInr.fasta protein database.
RNA isolation
Total RNA was extracted from freshly harvested cells
and from frozen tissues according to the protocol of
Chomczynski and Sacchi [25].
Real time PCR analysis
Real time PCR was performed in a Light Cycler (Roche,
Germany). All PCR experiments were performed using the
Light Cycler DNA Master SYBR Green I kit provided by
Roche Molecular Biochemicals (Mannheim, Germany).
Each reaction (20 lL) contained 2 lLcDNA,3.0m
M
MgCl
2
, 1 pmol of each primer and 2 lLofFastStarter

Mix (containing buffer, dNTPs, SYBR Green and hotstart
Taq polymerase). The primers used are summarized in
Table 1.
The amplification program consisted of one cycle at
95 °C for 10 min, followed by 40 cycles with a denaturing
phase at 95 °C for 15 s, an annealing phase of 5 s at 60 °C
and an elongation phase at 72 °C for 15 s. A melting curve
analysis was carried out after amplification to verify the
4516 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
accuracy of the amplicon. For verification of the correct
amplification, PCR products were analyzed on an ethidium
bromide stained 2% agarose gel.
In each real-time PCR run for each gene product under
investigation and for b-actin a calibration curve was
included, that was generated from serial dilutions (1 : 1,
1 : 10, 1 : 100, 1 : 1000) of a cDNA generated from the
pooled RNA of the normoxic (control) cultures (at the
different time points) of the respective experimental series
(standard cDNA). Analysis of the individual unknowns
therefore yielded values relative to this pool. Data are
presented as the relative mRNA/b-actin mRNA ratio. The
mRNA/b-actin mRNA ratio of the time standards (pools)
cDNA was set to 1.0 (i.e. normoxia, 21% oxygen). Data
are therefore expressed as relative values related to
normoxia.
Statistics
Levels of significance between groups were calculated using
ANOVA
test followed by Bonferoni’s reduction for multiple
comparisons. P < 0.05 was considered significant.

Results
Screening the rat vascular smooth muscle cell line A7r5 for
hypoxia (1% oxygen for 24 h) induced proteins by two-
dimensional electrophoresis revealed a highly reproducible
two- to fourfold up-regulated abundance of two proteins
(Fig. 1). One of them appeared with two slightly different
masses around 60 kDa on SDS/PAGE with a pI around
pH 5.7. Analysis of tryptic peptides by ESI-MS identified
these proteins as rat prolyl-4-hydroxylase aI subunit
(P4ha1). The slightly different molecular masses of the aI
subunit probably result from different glycosylation [26].
The second protein appeared as a single spot with a
somewhatsmallermolecularmassonSDS/PAGEthanthe
prolyl-4-hydroxylase aI subunit, but with a rather similar pI
of pH 5.7. This spot was identified by ESI-MS as rat prolyl-
4-hydroxylase aII subunit (P4ha2). Repeated analysis of
different gel runs using different protein extracts from A7r5
cells indicated that the prolyl-4-hydroxylase aI spot was
increased two to 2.5-fold and the prolyl-4-hydroxylase aII
spot was increased three- to fourfold after exposure of the
cells to 1% oxygen for 24 h.
Table 1. Primers used for real-time PCR of procollagen, prolylhydroxylases, PDI and lysylhydroxylases mRNAs.
Mouse (gi:836897) prolylhydroxylase alpha I
Sense:
5¢-CGGGATCCTAGACCGGCTAACAAGTA-3¢
Antisense:
5¢-GGAATTCCAAGCAGTCCTCAGCTGT-3¢
Rat (gi:474939) prolylhydroxylase alpha I
Sense:
5¢-CGGGATCCTCGGACACCCTGTAAATG-3¢

Antisense:
5¢-GGAATTCCAAGCAGTCCTCAGCTGT-3¢
Mouse (gi:6754969) prolylhydroxylase alpha II
Sense:
5¢-CGGGATCCTGCAGGCAGAATTCTTCA-3¢
Antisense:
5¢-GGAATTCCCAGTCTGTGTTCAACCG-3¢
Rat (gi: 6754969) prolylhydroxylase alpha II
Sense:
5¢-CGGGATCCTGCAGGCAGAATTCTTCA-3¢
Antisense:
5¢-GGAATTCGCTGAACTGAGAGGTTAG-3¢
Mouse (gi: 20913928) and rat (gi: 6981323) protein disulfide isomerase
Sense:
5¢-CGGGATCCAGCAGTATGGTGTCCGTG-3¢
Antisense:
5¢-GGAATTCACCGTCACTTCGCTTGAG-3¢
Mouse (gi: 6755105) lysylhydroxylase I
Sense:
5¢-AACTGGTGGCCGAGTGGG-3¢
Antisense:
5¢-GCAGGGTGTCATAGGCCA-3¢
Rat (gi: 409058) lysylhydroxylase I
Sense:
5¢-AACTGGTGGCCGAGTGGG-3¢
Antisense:
5¢-GCCCATTTCAAACTTGAG-3¢
Mouse (gi: 6755107) lysylhydroxylase II
Sense:
5¢-GCACATTGGGAAACGCTA-3¢

Antisense:
5¢-AATTTTGCATTTGTGATC-3¢
Rat (gi: 6755107) lysylhydroxylase II
Sense:
5¢-CCTGTTGTGCACATTGGG-3¢
Antisense:
5¢-AATTTTGCATTTGTGATC-3¢
Mouse (gi: 424103) procollagen Col1a1
Sense:
5¢-ACCTCAAGATGTGCCACT-3¢
Antisense:
5¢-TCCATCGGTCATGCTCTG-3¢
Rat (gi: 807263) procollagen Col1a1
Sense:
5¢-ACCTCAAGATGTGCCACT-3¢
Antisense:
5¢-GATGTACCAGTTCTTCTG-3¢
Mouse and rat (gi: 6671508) b-actin
Sense:
5¢-CGGGATCCCCGCCCTAGGCACCAGGGTG-3¢
Antisense:
5¢-GGAATTCGGCTGGGGTGTTGAAGGTCTCAAA-3 ¢
Ó FEBS 2003 Hypoxia and procollagen hydroxylases (Eur. J. Biochem. 270) 4517
To investigate the underlying mechanism for the
increased expression of the P4h subunits in response to
hypoxia, we next analyzed mRNA expression for P4ha1
and P4ha2 mRNA. Real-time PCR analysis revealed a
characteristic time-dependent increase of the mRNA abun-
dance in A7r5 cells incubated at 1% oxygen for P4ha1 and
P4ha2, starting around 4 h of hypoxia, the induction of

P4ha2 mRNA being stronger than that of P4ha1 (Fig. 2A).
In view of this concordant regulation, we further considered
the possibility that also the expression other hydroxylases
involved in collagen fiber formation might be regulated by
the cellular oxygen tension. In fact, it turned out that also
the mRNAs for lysyl hydroxylases I and II (PLOD1 and -2)
increased clearly during hypoxia, in a very similar fashion to
the mRNAs for the prolyl hydroxylases. In addition, also
the mRNA for protein disulfide isomerase (PDI), as the
b-subunit of prolyl hydroxylases, increased in A7r5 cells,
although significantly delayed and to a lesser extent. After
24 h of hypoxia the mRNA abundance was five-, 12-, six,
seven- and fivefold increased for P4ha1, P4ha2, PDI,
PLOD1 and PLOD2, respectively. Notably the abundance
of procollagen Ia was not changed by hypoxia (Fig. 2B).
Very similar results to those with hypoxia were obtained,
when A7r5 cells were incubated with the iron-chelator
deferoxamine (100 lmolÆL
)1
) at ambient oxygen tension
(21% O
2
). After 24 h mRNA abundance was increased
five-, 16-, two-, five- and 10-fold, for P4ha1, P4ha2, PDI,
PLOD1 and PLOD2, respectively (Fig. 3A), whilst the
mRNA abundance for procollagen Ia was unchanged.
Also, cobalt(II) chloride (100 lmolÆL
)1
) moderately
increased the mRNAs of the hydroxylases, but not of

procollagen Ia mRNA (Fig. 3B).
To test for the cell and species specificity of the changes of
the enzyme expression in response to hypoxia, we also
analyzed the mouse juxtaglomerular cell line As4.1. As we
have recently found that these cells respond to hypoxia
rather rapidly [27], we exposed the cells to either hypoxia
(0.5% oxygen) or deferoxamine (100 lmolÆL
)1
) for only
4.5 h and assayed the mRNA levels of the procollagen
hydroxylases. By these maneuvers, P4ha1 mRNA increased
five- to eightfold, P4ha2 mRNA 25- to 33-fold, PDI mRNA
twofold, PLOD1 mRNA fivefold, and PLOD2 mRNA
twofold (Fig. 4).
As the combination of the stimulatory effects of hypoxia,
deferoxamine and cobalt suggested a possible involvement
of the hypoxia-inducible transcription factor (HIF) in the
activation of P4h, and PLOD gene expression by hypoxia,
we further examined the expression of these genes in a cell
line with a defective HIF, namely the murine hepatoma cell
line Hepa1C4. In the control cell line, Hepa 1 both hypoxia
(0.5% oxygen) and deferoxamine (100 lmolÆL
)1
) clearly
induced P4ha1 (four- and sevenfold) and P4ha2 mRNA
(seven- and fourfold) and to a lesser extent also PDI mRNA
(two- and 1.5-fold), whilst procollagen Ia mRNA remained
unchanged after 12 h of stimulation (Fig. 5A). The stimu-
latory effect of hypoxia and of deferoxamine on P4ha1 and
P4ha2 mRNA and PDI mRNA was almost abrogated in

Hepa1C4 cells (Fig. 5B), supporting the assumption that
the expression of these genes was driven by HIF. Unfor-
tunately, mRNA levels for the PLOD mRNAs were too low
to allow reasonable semiquantification in both Hepa1 and
Hepa1C4 cells.
This first indication about an essential role of HIF in the
triggering of prolylhydroxylase gene expression was further
Fig. 1. Two-dimensional electrophoresis of proteins isolated from the rat vascular smooth muscle cell line A7r5. The indicated protein spots were
up-regulated by exposure of the cells to hypoxia (1% O
2
) for 12 h.
4518 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
corroborated in mouse embryonic fibroblasts lacking the
HIF-1a subunit. As shown in Fig. 6 hypoxia, deforoxamine
and cobalt stimulated the expression of P4ha1, P4ha2, PDI,
PLOD1 and PLOD2 mRNAs in wild-type embryonic
fibroblasts, but not in embryonic fibroblasts lacking the
HIF-1a subunit.
As the enzymes involved in collagen formation are
important for the correct folding of the protein, it is in
principle conceivable, that their expression is also triggered
Fig. 3. P4ha1, P4ha2, PDI and PLOD1, PLOD2, and procollagen
Ia mRNA in A7r5 cells after exposure to cobalt(II) chloride
(100 lmolÆL
-1
) (A) or to deferoxamine (100 lmolÆL
-1
) (B) for 12 h at
21% O
2

. Data are means ± SEM of five experiments each. *P <0.05
vs. control (21% O
2
). Controls are the means of five experiments and
the average mRNA/b-actin mRNA ratio is set to 1 (dotted line).
Fig. 4. P4ha1, P4ha2, PDI, PLOD1 and PLOD2 mRNA in mouse
As4.1 cells after exposure to hypoxia (0.5% O
2
), to deferoxamine
(100 lmolÆL
-1
) and to cobalt(II) chloride (100 lmolÆL
-1
)(at 21%O
2
)for
4.5 h of incubation. Data are means ± SEM of five experiments each.
*P < 0.05 vs. control (21% O
2
). Controls are the means of five
experiments and the average mRNA/b-actin mRNA ratio is set to 1
(dotted line).
Fig. 5. P4ha1, P4ha2, PDI mRNA and procollagen Ia mRNA in Hepa1
(A) and in Hepa1C4 cells (B) after exposure to hypoxia (0.5% O
2
)orto
deferoxamine (100 lmolÆL
-1
)at21%O
2

. Data are means ± SEM of
five experiments each. *P <0.05 vs. control (21% O
2
). Controls are
the means of five experiments and the average mRNA/b-actin mRNA
ratio is set to 1 (dotted line).
Fig. 2. Time course of P4ha1, P4ha2 mRNA (A) and PLOD1, PLOD2
mRNA (B) and PDI, procollagen Ia mRNA (C) in A7r5 cells after
exposure of the cells to 1% O
2
. Data are means ± SEM of five
experiments. *P < 0.05 hypoxia (1% O
2
) vs. normoxia (21% O
2
).
Controls are the means of five experiments and the average mRNA/b-
actin mRNA ratio is set to 1 (dotted line).
Ó FEBS 2003 Hypoxia and procollagen hydroxylases (Eur. J. Biochem. 270) 4519
by a disturbance of protein folding due to energy depletion
in the course of cellular hypoxia. This so-called unfolded
protein response (UPR) can also be elicited by tunicamycin
at normal oxygen tensions [28], as shown in Fig. 7.
Tunicamycin gave a clear twofold increase of PDI mRNA,
a more moderate increase of the mRNAs for prolyl
hydroxylases, and did not increase the mRNA abundance
of the other enzymes.
Discussion
Our data show that the expression of a functional cluster of
hydroxylases enzymes crucially required for procollagen

maturation and collagen fiber formation are regulated in
concert by the tissue oxygen tension, in the way that they are
up-regulated at low oxygen tensions, i.e. hypoxia. This
observation thus confirms the previous notion about an
increased prolyl hydroxylase activity in hypoxic tissues
in situ [10], as well as the up-regulation of P4ha1 mRNA and
protein in response to hypoxia in cell culture [14]. As to the
induction of P4ha1 mRNA and protein our data obtained
in A7r5 cells are almost identical with regard to time course
and to ability to stimulate to those reported by Takahashi
and coworkers for fetal lung fibroblasts [14]. As to the
induction of PDI mRNA, our data are also in good
accordance regarding the delayed time course and the
differential ability to stimulate by hypoxia, iron chelation
and cobalt with those reported by Tanaka and coworkers
for cultured astrocytes [15]. Our findings thus on the one
hand fully support these previous findings and on the other
hand extend them by far and set them in a more general
context, as both a-subunits of P4h, PDI as the b-subunit of
P4h [13] and the collagen procollagen lysyl hydroxylases
PLOD1 and PLOD2 are oxygen regulated. As these effects
were seen in different mouse and rat cell lines, we infer
that the up-regulation of the procollagen hydroxylases
during hypoxia is a more general effect with physiological
relevance.
Such a concerted regulation of these key enzymes of
collagen formation appears reasonable from a physiological
point of view, as all of these enzymes use oxygen directly as
a common substrate and because the formation of collagen
requires the coordinated activities of all the enzymes. Such a

common regulation of a functional cluster of genes by the
oxygen tension has already been found for the enzymes
involved in the glycolytic cascade [29] and also for the key
players of angiogenesis [30].
Considering the parallel up-regulation of enzymes
involved in collagen fiber formation raises the question of
whether this up-regulation is physiologically primarily
meant to increase collagen formation in hypoxic tissues,
or if it reflects more a compensatory change of the
concentration of enzyme molecules to maintain a given
normal hydroxylation rate at altered substrate (oxygen)
concentrations (Fig. 8). The explanation as a compensatory
increase of gene expression was previously also presented
for the increased expression of the endoplasmic oxido-
reductase Ero1-L during hypoxia which transfers oxidizing
equivalents onto PDI [27]. Such a view would be supported
by the observation that the expression of the procollagen
(Ia) gene itself was not regulated by the oxygen tension,
which is also in accordance with data obtained by others [7].
As the genes for P4ha1, P4ha2, PDI, and PLOD 1,2 are
localized on different chromosomes the question arises
concerning the mechanisms underlying the orchestrated
expression of these enzymes by the oxygen tension.
The findings that the effect of hypoxia on the gene
expression of the hydroxylases was mimicked by the iron
Fig. 6. P4ha1, P4ha2, PDI, PLOD1 and PLOD2 mRNA in mouse
embryonic fibroblasts with intact and with disrupted HIF-1a gene after
exposure to hypoxia (0.5% O
2
) or to deferoxamine (100 lmolÆL

-1
)at
21%O
2
for 24 h. Data are means ± SEM of five experiments each.
*P < 0.05 vs. control (21% O
2
). Controls are the means of five
experiments and the average mRNA/b-actin mRNA ratio is set to 1
(dotted line).
Fig. 7. P4ha1, P4ha2, PDI, PLOD1, PLOD2, and procollagen I(a)
mRNA in mouse As4.1 cells after incubation with tunicamycin
(10 lgÆmL
-1
) for 24 h at 21% O
2
. Data are means ± SEM of five
experiments each. *P < 0.05 vs. control (21% O
2
). Controls are the
means of five experiments and the average mRNA/b-actin mRNA
ratio is set to 1 (dotted line).
4520 K H. Hofbauer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
chelator deferoxamine and the divalent cation cobalt,
suggest that these genes are under the control of the
hypoxia inducible transcription factor HIF. HIF is a
heterotetramer consisting of an a-andab-subunit [31].
The protein abundance of this protein dimer is inversely
related to the cellular tension, because the a- but not
b-subunit protein stability is dependent on the oxygen

tension, in the way that the a-subunit is more stable at low
oxygen tensions. The reason for this behavior is an oxygen
dependent prolyl-hydroxylation of the a-subunit, which
finally directs the protein to proteasomal degradation [32].
The assumption that HIF could in fact be a main trigger of
the procollagen hydroxylases is further corroborated by the
findings that the stimulatory effect of hypoxia on gene
expression was absent in cells with a functional inactive HIF
or lacking the HIF-1a protein in general. In fact, for the
P4ha1I the involvement of HIF in the activation of gene
expression during hypoxia has recently been directly dem-
onstrated [14]. A search for the HIF-binding consensus
sequence CGTG revealed six, six, two, nine and 10
theoretical HIF binding sites within the first 1 kb of the
5¢-promoter region of mouse P4ha1, P4ha2, PLOD1,
PLOD, and PDI, respectively.
Although prolyl hydroxylation is a critical event for both
procollagen triple helix stabilization on the one hand and
for the stability of HIF-1a protein, different prolyl
hydroxylases appear to be required for these processes.
HIF-a prolyl hydroxylation is managed by PHD-1, -2 and
-3 [33,34], whilst procollagen prolylhydroxylation is
performed by P4h [13], which does not accept HIF-a as
a substrate [31]. Interestingly, the expressions of PHD-3
[35,36] and eventually of PHD-2 itself are also oxygen
sensitive [35,36]. They are up-regulated by hypoxia by a
process critically involving HIF, which in turn is also the
substrate of PHD-2 and PHD-3. Thus, PHD-2, PHD-3 and
P4h expression appear to be subject to a common control by
oxygen, whilst the expression of PHD-1 is not. The

physiological meaning of this differential regulation of
PHD-gene expression remains to be clarified.
In spite of the similar regulation of PHD-2, PHD-3 and
P4h expression by oxygen, not only the protein target but
also the intracellular localization is different between the
two groups of enzymes. Whilst PHD-1, -2 and -3 are
mainly cytosolic and nuclear proteins [37], P4h is like the
lysyl hydroxylases PLOD1 and -2, being localized within
the endoplasmic reticulum [13,38]. This localization could
be of some interest, as HIF-regulated genes, as identified
so far, encode mainly for cytosolic or for secreted proteins
[28,39]. Constituents of the endoplasmic reticulum as
being HIF-regulated have not yet been frequently reported
[39]. We have recently obtained evidence that the expres-
sion of endoplasmic oxidoreductase Ero1-L, which oxid-
izes PDI, is also controlled by HIF [27]. Given the
conjunction of PDI with PDH-4 and the conjunction of
PDI with Ero1-L as an essential oxidizing enzyme of PDI,
there arises the concept a network of endoplasmic
enzymes that mediate oxygen dependent reactions and
that are in turn regulated by the oxygen tension in an
inverse fashion most likely regulated by the transcription
factor HIF-1.
It must be considered in this context that the expression
of endoplasmic proteins with folding or chaperone function
might also be indirectly triggered by cellular hypoxia
through the UPR [40] induced by cellular energy depletion
[41,42]. We have addressed this issue therefore by investi-
gating the influence of the UPR for the expression of the
procollagen hydroxylases. With the exception of PDI, which

is known to be induced by the UPR [41], however, none of
the other enzymes was relevantly induced by the UPR,
supporting the assumption that they are more directly
triggered by HIF.
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