Results Expression of ET-receptor subtypes by human primary dermal fibroblasts Cell surface binding of [125I]1 revealed specific ET-1 binding sites on human primary dermal fibroblasts.. Ad
Trang 1and TGF-b1
Angelika Horstmeyer1, Christoph Licht2, Gabriele Scherr1, Beate Eckes1and Thomas Krieg1
1 Department of Dermatology and 2 Children’s Hospital, University of Cologne, Cologne, Germany
Connective tissue homeostasis requires the tightly
coordinated regulation of synthesis and degradation of
extracellular matrix constituents (ECM) This involves
a number of different factors including signalling of
cytokines in an auto- and paracrine manner, e.g
trans-forming growth factor-b1 (TGF-b1), platelet-derived
growth factor (PDGF), basic fibroblast growth factor
(bFGF) and tumour necrosis factor-a (TNF-a)
Collagen I is the major fibrillar collagen in the der-mal connective tissue and comprises approximately 80% of the total amount of the collagens synthesized
by fibroblasts Dysregulated collagen I synthesis at any step leads to diseases such as scurvy or fibrosis of skin and internal organs
TGF-b1 transmits its biological effects via serine-threonine receptors and Smad-proteins [1] Some of
Keywords
Connective tissue growth factor; dermal
fibroblast; endothelin receptor subtypes;
fibrosis; phospholipase
Correspondence
A Horstmeyer, Department of Dermatology,
University of Cologne,
Joseph-Stelzmann-Str 9, D-50931 Cologne, Germany
Fax: (+)49 221 478 5949
Tel: (+)49 221 478 6152
E-mail: ahorstmey@gmx.de
(Received 29 May 2005, revised 7 October
2005, accepted 13 October 2005)
doi:10.1111/j.1742-4658.2005.05016.x
Endothelin-1 (ET-1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC) ET-1 signals are transduced by two receptor subtypes, the ETA- and ETB-receptors which activate different Ga proteins Here, we investigated the expression of both ET-receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA-receptor is the major ET-receptor subtype expressed To determine further signalling intermedi-ates, we inhibited Gai and three phospholipases Pharmacologic inhibition
of Gai, phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase
D (PLD), but not of phospholipase Cb, abolished the increase in collagen I
by ET-1 Inhibition of all phospholipases revealed similar effects on TGF-b1 induced collagen I synthesis, demonstrating involvement of PC-PLC and PLD in the signalling pathways elicited by ET-1 and TGF-b1 ET-1 and TGF-b1 each stimulated collagen I production and in an additive manner ET-1 further induced connective tissue growth factor (CTGF), as did TGF-b1, however, to lower levels While rapid and sustained CTGF induction was seen following TGF-b1 treatment, ET-1 increased CTGF in
a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET-1 treatment Coincidentally at
5 days of permanent ET-1 stimulation, a switch in ET-receptor subtype expression to the ETB-receptor was observed We conclude that the signal-ling pathways induced by ET-1 and TGF-b1 leading to augmented collagen
I production by fibroblasts converge on a similar signalling pathway Thereby, long-time stimulation by ET-1 resulted in a changed ET-receptor subtype ratio and in a biphasic CTGF induction
Abbreviations
bFGF, basic fibroblast growth factor; CTGF, connective tissue growth factor; DAG, 1,2-diacylglycerol; ECM, extracellular matrix; ET-1, Endothelin-1; ET-receptor, endothelin receptor; FBS, fetal bovine serum; IP 3 , inositol triphosphate; Ga, Ga protein; MMP, matrix
metalloproteinase; PC, phosphatidylcholine; PDGF, platelet-derived growth factor; PKC, protein kinase C; PLC, phospholipase; PTX, Pertussis toxin; TGF-b1, transforming growth factor-b1; TIMP-1, tissue inhibitory matrix metalloproteinase-1; TNF-a, tumour necrosis factor-a.
Trang 2the actions of TGF-b1 are mediated by the induction
of connective tissue growth factor (CTGF) [2], which
stimulates collagen production by fibroblasts [3]
CTGF is coexpressed with TGF-b1 at sites of tissue
fibrosis in numerous tissues [4]
Besides TGF-b1 and CTGF, the peptide hormone
endothelin-1 (ET-1) was also reported to be involved
in the pathophysiology of cardiac [5,6], renal [7],
pul-monary [8–10], and dermal fibrosis such as systemic
sclerosis⁄ scleroderma [11–13] Serum concentrations of
ET-1 as well as TGF-b1 were increased in scleroderma
patients [8,14,15] These patients showed changes in
total ET-receptor expression with an increased
percent-age of the ETB-receptor, one of the two ET-receptor
subtypes [11,12,15]
An antagonist against both ET-receptor subtypes
abolished ET-1 induced synthesis of matrix proteins
such as fibronectin and collagens I and III, suggesting
that their synthesis is mediated via both ET-receptor
subtypes [11] In contrast, other authors reported that
these effects were solely mediated via ETA-receptor
stimulation [5] Besides these inducing effects,
ETA-receptor stimulation also contributes to ECM
degra-dation by reducing expression of tissue inhibitor of
matrix metalloproteinase-1 (TIMP-1) and inducing
that of matrix metalloproteinase-2 (MMP-2) [7,16],
which in turn degrades MMP-1 cleaved collagen I [17]
Regulation of MMP-1 expression by ET-1 has been
discussed controversially demonstrating repression as
well as induction [11,18] However, the opposing
effects of ET-1 on collagen I synthesis and breakdown
clearly indicate an important role for ET-1 in the
dynamic regulation of ECM and its dysregulation in
disease [19]
ET-1 and its isoforms ET-2 and )3 belong to a
peptide hormone family, which was originally
charac-terized as a vasoconstrictor in the systemic and
pulmonary circulation [15] Since then, apart from
regulating ECM deposition and turnover [7,11,18,19],
ET-1 was further shown to induce differentiation [13],
mitogenesis [11] or release of pro-inflammatory
cyto-kines such as the ECM regulatory cytocyto-kines TGF-b1
[20,21], PDGF, bFGF [22] and TNF-a [23]
The endothelins exert their biological effects via two
specific cell surface receptor subtypes, the ETA- and the
ETB- receptor, which are members of the G
protein-coupled receptor family The ETA-receptor couples to
heterotrimeric Ga proteins such as Gaq, Gas, Ga12⁄ 13
[24–26] and in some cell types to Gai [27–29], whereas
the ETB- receptor couples to Gai and Gaq [25] Signal
transduction triggered by binding of one of the three
endothelins leads to changed concentrations of various
second messengers such as Ca2+, 1,2-diacylglycerol
(DAG), inositol triphosphate (IP3), cyclic adenosine 3¢,5¢-monophosphate (cAMP) and arachidonic acid [24,27]
The induction of collagen I synthesis by ET-1 was described to involve protein kinase C-d (PKC-d), p44⁄ 42 MAPK (pERK1⁄ 2) and SP-1 [30,31] PKC-d is a mem-ber of the novel PKCs Their activation depends exclu-sively on DAG, in contrast to the conventional PKCs, which require DAG and Ca2+in addition to phospha-tidylserine for activation [32] ET-1 increases cellular DAG concentrations derived from 4,5-bis-phosphate (PIP2) by PIP2-PLC-b [24] and phosphatidylcholine (PC) by PLD [33–35] or by PC-PLC activation [34–36]
To better understand the molecular mechanisms underlying the role of ET-1 in regulating ECM depos-ition and its involvement in fibrotic processes, we analysed the signalling pathway inducing collagen I synthesis between the cell surface receptor and PKC-d
by pharmacological intervention using specific inhibi-tors We also investigated the effects of combined ET-1 and TGF-b1 stimulation and of prolonged stimu-lation with respect to ET-receptor subtype expression and the induction of CTGF and TGF-b1 transcripts
Results Expression of ET-receptor subtypes by human primary dermal fibroblasts
Cell surface binding of [125I]1 revealed specific
ET-1 binding sites on human primary dermal fibroblasts Binding was confirmed by displacing labelled ligand by specific ET-receptor antagonists including the nonsub-type specific antagonist PD156252, the ETA-receptor selective antagonist BQ123 and the ETB-receptor selective antagonist BQ788 The IC50 values were 5.4· 10)10 mfor BQ123, and 3.8· 10)7mfor BQ788, clearly demonstrating high affinity binding to the ETA-receptor (Fig 1A) These data are in agreement with previously published IC50values [37,38]
Results from surface binding analysis were further confirmed by immunoblotting (Fig 2A) In total cell lysates, the ETA-receptor was clearly detected as well
as in membrane fractions The ETA-receptor was enriched in membrane fractions as revealed by two specific bands, a predominant band of approximately
55 kDa and a less prominent one of approximately
45 kDa as described [24,39,40] Using immunodetec-tion, we were unable to show ETB-receptor expression
in fibroblasts However, applying RT-PCR, both, the ETA- as well as the ETB-receptor were detectable as single bands at 135 bp (ETA-receptor) and 150 bp (ETB-receptor; Fig 2B)
Trang 3We therefore conclude that the ETA-receptor is the receptor predominantly expressed by human primary dermal fibroblasts, while ETB-receptor expression lev-els are much lower as reported lev-elsewhere [11]
Additive induction of collagen I levels by ET-1 and TGF-b1
ET-1 stimulation (100 nm) induced a twofold increase
in the synthesis of collagen I compared to unstimu-lated controls (Fig 3A), in concordance with published data [11,13] By immunoblotting, collagen I was detec-ted by two different antibodies against human collagen
I, a polyclonal and a monoclonal one (data not shown) Both antibodies recognized collagen I derived from fibroblast culture supernatants as well as purified collagen I As a positive control, cultures stimulated
by 180 pm TGF-b1 [41,42] were included in all subse-quent analyses Induction of collagen I by TGF-b1 and by ET-1 was perceptible by coomassie staining as well as by immunoblotting (Fig 3B) The immunoblot thus confirms the specifity of the coomassie staining (see Experimental procedures)
Comparing collagen I levels induced by either ET-1 or TGF-b1 or a combination of both revealed that either hormone alone enhanced collagen I levels
in fibroblasts compard to unstimulated cells (control) and that the combination induced an additive effect Fibroblasts responded to stimulation by increasing collagen I synthesis in the order: unstimulated
Fig 2 Expression of ET-receptors (A) Immunoblotting: human primary dermal fibroblasts were cultured for two days and cells were lysed
or fractionated Samples were separated by 10% SDS ⁄ PAGE and electrophoretically transferred to nitrocellulose ET-receptors were visual-ized with polyclonal rabbit anti-ETA- or anti-ETB-receptor antibody Lanes: membrane fraction (membrane), total cell lysate (lysate) The immunoblot shown represents three independent experiments, each performed with cells from different donors and cell isolations (B) RT-PCR: human primary dermal fibroblasts were cultured for one day, then starved for one day RNA was isolated, reverse-transcribed and subjected to PCR in presence of specific primer pairs for the ETA- or ETB-receptor Samples were separated by 3% agarose-ethidium bro-mide gel electrophoresis Results from two different donors (lane 1, 2) and negative controls (H2O) are shown The gel shown represents four independent experiments, performed with cells from different donors and cell isolations.
Fig 1 Displacement of [ 125 I]ET-1 by specific ET-receptor
antago-nists Human primary dermal fibroblasts were cultured on 96 well
plates for two days Fibroblasts were incubated with [125I]ET-1
(25 p M ) with antagonists as indicated, lysed, and bound ligand was
determined using a c-counter (A) Results are summarized as IC50
values and means ± SEM of six independent experiments each
performed with cells from different donors and cell isolations (B)
Representative displacement curves of the three antagonists:
PD156252 (n), BQ123 (m), BQ788 (d) Results are means ± SEM.
Trang 4control < ET-1 < TGF-b1 < ET-1 + TGF-b1
(Fig 3B)
Analysis of ET-1 and TGFb1 signalling
mechanisms resulting in collagen I synthesis
To determine the signalling pathway between the
ET-receptor and its cognate PKC-isoform, that mediates
the ET-1 induced collagen I synthesis in fibroblasts,
specific inhibitors were applied Cadwallader et al [29]
described Gai-dependent ET-1 stimulation of PKC via
the ETA-receptor in rat fibroblasts We therefore
inves-tigated the involvement of Gai in human primary
dermal fibroblasts Pertussis toxin (PTX), a specific Gai
inhibitor [26], strongly decreased ET-1-induced collagen
I synthesis But PTX did not alter unstimulated, basal
collagen I synthesis (Fig 4) as previously described
[43] Further, collagen I levels induced by TGF-b1
stimulation were also not altered by PTX, as described [43] This indicated involvement of Gai leading to enhanced collagen I levels after ET-1 stimulation
To determine the phospholipases responsible for PKC activation [32], we examined the possible DAG donors PLC-b, PC-PLC and PLD PLCb, a down-stream effector of the Gaq [44] as well as of the bc subunit of Gai [45] was inhibited specifically by U73122 [46] Inhibiting PLC-b by U73122 did not alter ET-1-induced collagen I synthesis, nor did it influence basal or TGF-b1-stimulated collagen I levels (Fig 5A) Next, we analysed the involvement of the PC-phospho-lipases We used D609 to specifically inhibit PC-PLC [46], and neomycin [47–49] and N-butanol [50–52] as specific inhibitors of PLD The inhibition of PC-PLC
by D609 (Fig 5B) and inhibition of PLD by neomycin (Fig 5C) as well as by N-butanol (not shown) abol-ished the increase in collagen I levels after ET-1
A
B
Fig 3 ET-1 induces collagen I synthesis Human primary dermal fibroblasts were cultured without or with hormone (100 n M ET-1, 180 p M TGF-b1) for 2 days The entire cell culture media were incubated with pepsin, TCA-precipitated and separated by 6% SDS ⁄ PAGE (A) Pro-teins were electrophoretically transferred to nitrocellulose and collagen I was visualized with polyclonal rabbit anticollagen I antibody Lanes: unstimulated fibroblasts (control), fibroblasts stimulated with ET-1 (ET-1), fibroblasts stimulated with TGF-b1 (TGFb1), one lg of purified colla-gen I (positive control) The histogram represents densitometric ratios of (stimulated: unstimulated) collacolla-gen I signals from three independent experiments, performed with cells from different donors and cell isolations Unstimulated values were set as 1.0 Results are means ± SEM Asterisks represent statistically significant differences, P < 0.05 (B) Two specific collagen I bands representing the a1 and a2 chains
of collagen I were stained by Coomassie Blue Lanes: unstimulated fibroblasts (control), fibroblasts stimulated with ET-1 (ET-1), fibroblasts stimulated with TGF-b1 (TGFb1), fibroblasts stimulated with ET-1 and TGF-b1 (ET-1 TGFb1) The histogram represents densitometric ratios (stimulated: unstimulated) of collagen I signals from four independent experiments, performed with cells from different donors and cell iso-lations Unstimulated values were set as 1.0 Results are means ± SEM.
Trang 5stimulation Of note, simultaneous PLD inhibition and
ET-1 stimulation significantly reduced collagen I levels
to below basal values TGF-b1 signalling was affected
similarly, with no effect by inhibiting PLC-b (Fig 5A),
strong down-regulation of collagen I levels after
inhi-bition of PC-PLC (Fig 5B), and similar collagen I
down-regulation after PLD inhibition (Fig 5C) These
results demonstrate a crucial role for the
PC-phospho-lipases in collagen I induction by both ET-1 and
TGF-b1 Our results are in agreement with previous reports
that demonstrated the involvement of PC-PLC in
TGF-b1-mediated regulation of other genes not
inclu-ding collagen I in further cell types [53,54]
Long-term stimulation by ET-1 and TGF-b1
Increased serum concentrations of ET-1 and TGF-b1
were shown to be present in sera of patients with
fibro-tic diseases [8,14,15] In addition, it has been described
that TGF-b1 causes a persistent increase in collagen I
[41] partly via CTGF gene activation [55] It is thereby
reported for both hormones that an induced CTGF
gene expression correlates with an increased amount of
the CTGF hormone [18,56] To clarify whether ET-1
also mediates its effects via CTGF, we stimulated
fibroblasts with ET-1 and TGF-b1 for five days and
examined CTGF and TGF-b1 gene activation
(Fig 6A) ET-1 stimulation resulted in a biphasic
induction of CTGF We observed a weak and short
initial increase of CTGF transcripts three hours after
ET-1 stimulation, which thereafter decreased to basal
levels However, a stimulation for five days resulted in
a second, stronger increase in CTGF RNA levels In contrast to ET-1, stimulation with TGF-b1 led to per-sistent induction of CTGF transcripts during the entire stimulation period
As ET-1 induced TGF-b1 RNA expression in var-ious cell types [20,21] and both, ET-1 and TGF-b1 act additionally to induce collagen I synthesis (Fig 3B) we examined whether ET-1 induces TGF-b1 gene activa-tion in human primary dermal fibroblasts In contrast
to TGF-b1, that induced its own gene activation, ET-1 stimulation failed to induce TGF-b1 RNA levels (Fig 6B) This indicates a TGF-b1 independent action
of ET-1
Previous reports described altered ratios of ETA- to ETB-receptors in fibroblasts cultured from the skin of patients with systemic sclerosis [11,12] We were inter-ested to see whether long-term in vitro stimulation of fibroblasts from healthy donors with ET-1 also leads
to similar modulations Indeed after stimulation with ET-1 over five days we observed induced levels of the ETB-receptor, while ETA-receptor levels were strongly diminished (Fig 7C)
Discussion Using human primary dermal fibroblasts, the ETA-receptor was clearly detectable by immunoblotting in contrast to the ETB-receptor, which required more sensitive investigation by RNA analysis for detection Furthermore, ET-1 displacement studies with subtype-specific antagonists confirmed binding only to the ETA-receptor Thus, the ETA-receptor is the main
Fig 4 Analysis of signalling mechanism: Gai After pretreatment with PTX (100 ngÆmL)1, overnight) human primary dermal fibroblasts were cultured without or with hormone (100 n M ET-1, 180 p M TGF-b1) for 2 days Entire cell culture media were incubated with pepsin, TCA-preci-pitated and separated by 6% SDS ⁄ PAGE Two specific collagen I bands representing the a1 and a2 chains of collagen I were stained by Coomassie Blue Lanes: unstimulated fibroblasts (control), fibroblasts stimulated with (ET-1), fibroblasts stimulated with TGFb1 The histo-gram represents densitometric ratios (stimulated: unstimulated) of collagen I signals from three independent experiments, performed with cells from different donors and cell isolations Unstimulated values were set as 1.0 Results are means ± SEM Asterisks represent statisti-cally significant differences, P < 0.05.
Trang 6ET-receptor subtype expressed in human primary
der-mal fibroblasts, consistent with findings by Shi-wen
et al [11]
Interestingly, increased ET-1 serum concentrations
and a change in the ratio of the ETA- to the
ETB-receptor with an increase of the ETB-ETB-receptor on
fibro-blasts from patients with systemic scleroderma have
been described [11,12] This is in agreement with a
change in ET-receptor subtype expression pattern
towards the ETB-receptor after long-term stimulation
of five days by ET-1 The receptor subtype shift
towards the ETB-receptor described here is different
from cardiac and pulmonary fibrosis, where increased
numbers of ETA-receptor binding sites were observed
[9,57] A similar receptor subtype shift towards the
ETB-receptor is described in smooth muscle cells under
the development of arteriosclerosis [58] and for
endo-thelial cells under the condition of hypertension [59]
A changed ratio of ET-receptor subtypes results in
a modified signal transduction, because ETA- and
ETB-receptors couple in part to different Ga proteins For example in smooth muscle cells, a shift towards the ETB-receptor causes a deformation of these cells [60], whereas in human myocardial fibroblasts a shift towards the ETA-receptor profoundly interferes with cellular Ca2+ regulation [57] Moreover, it is described that the ETA-receptor regulates proliferation by pro-and antimitogenic actions [24,61] In addition, the ETA-receptor is a regulator of collagen I homeostasis
by inducing both, its synthesis and its degradation [7,16] in contrast to the ETB-receptor that only indu-ces collagen I synthesis [11,62] A changed ET-receptor subtype relation towards ETB-receptor thus may dis-turb the balance between synthesis and degradation of collagen I by ET-1 In this context, an increased num-ber of ETB-receptors may in turn be responsible for a change of signalling properties via different Ga coup-ling induced by ET-1 [63] This may be potentiated by
a changed internalization of ETB-receptors Homo-dimers and monomers of the ETB-receptor are
A
B
C
Fig 5 Analysis of signalling mechanism: DAG mediators PLC-b, PC-PLC and PLD After pretreatment with specific phospho-lipase inhibitors (15 min) human primary der-mal fibroblasts were cultured without or with hormone (100 n M ET-1, 180 p M TGF-b1) for two days Entire cell culture media were incubated with pepsin, TCA-precipita-ted and separaTCA-precipita-ted by 6% SDS ⁄ PAGE (A) PLCb inhibition by U73122 (10 l M ) (B) PC-PLC inhibition by D609 (185 l M ) (C) PLD inhibition by neomycin (500 l M ) Two specific collagen I bands representing the a1 and a2 chains of collagen I were stained by Coomassie Blue Lanes: unlated fibroblasts (control), fibroblasts stimu-lated with ET-1 (ET-1), fibroblasts stimustimu-lated with TGF-b1 (TGFb1) The histograms repre-sent densitometric ratios (stimulated: unstimulated) of collagen I signals from five independent experiments, performed with cells from different donors and cell isola-tions Unstimulated values were set as 1.0 Results are means ± SEM Asterisks repre-sent statistically significant differences,
P < 0.05.
Trang 7lysosomally degradated after ET-1 binding [64,65].
Elevated numbers of ETB-receptors increase the
prob-ability of heterodimerization between ETA- and
ETB-receptor proteins These heterodimers are recycled
after ligand binding as is the case for the monomeric
ETA-receptor [66] This additionally increases amount
of ETB-receptors present at the cell membrane It is
described [67] that the internalization and subsequent
lysosomal degradation of the ETB-receptor is
respon-sible for its short-term signalling period after ligand
binding Therefore, this changed ETB-receptor
inter-nalization may lead to a prolonged signal transduction
period via the ETB-receptor
The augmented autoinduction of ET-1 synthesis and
secretion via increased ETB-receptor numbers [68]
may also augment the intracellular signalling effects of
ETB-receptors This may result into an overshooting collagen I synthesis by ET-1 This is supported by our results demonstrating CTGF gene activation after ET-1 stimulation: ET-1 led to an activation of the early response gene CTGF at two time-points The first peak in CTGF gene activation was observed only three hours after ET-1 stimulation, whereas a second one was detectable after continuous stimulation for five days by ET-1 Reports from several groups indica-ted that induction of CTGF mRNA by ET-1 is regu-lated in a cell type-specific manner In human pulmonary fibroblasts, CTGF RNA was strongly induced by ET-1 for more than 12 hours [18], while in cardiac myocytes, CTGF was transiently induced for barely four hours, while in cardiac fibroblasts, no CTGF induction was observed [69]
A
B
Fig 6 Long-term effects of CTGF and
TGFb1 gene activation by ET-1 and TGF-b1.
Human primary dermal fibroblasts were
cul-tured without or with hormone (100 n M
ET-1, 180 p M TGF-b1) for the indicated
times Specific CTGF RNA or TGF-b1 RNA
were detected by nothern hybridization with
32 P labelled probes and visualized on X-ray
films (A) CTGF gene activation: samples are
unstimulated fibroblasts (C), fibroblasts
stimulated with ET-1 (E), fibroblasts
stimula-ted with TGF-b1 (T) Top panel: CTGF
detection, bottom panel: 18S detection
(loading control) The histograms represent
densitometric ratios of CTGF to 18S signals
from three to seven independent
experi-ments, performed with cells from different
donors and cell isolations Unstimulated
values were set as 1.0 Results are
means ± SEM Asterisks represent
statisti-cally significant differences, P < 0.05 (B)
TGF-b1 gene activation: samples are
unstimulated fibroblasts (C), fibroblasts
stimulated with ET-1 (E), fibroblasts
stimula-ted with TGF-b1 (T) Top panel: TGF-b1
detection, bottom panel: 18S detection
(loading control) The histograms represent
densitometric ratios of TGF-b1 signals
normalized to 18S signals from three to
seven independent experiments, performed
with cells from different donors and cell
isolations Unstimulated values were set as
1.0 Results are means ± SEM Asterisks
represent statistically significant differences,
P < 0.05.
Trang 8Our results indicate that in human primary dermal
fibroblasts ET-1 may mediate induced synthesis of
col-lagen I by activation of CTGF, which is not detectable
in unstimulated adult dermal fibroblasts [14] Upon
activation by TGF-b1, CTGF acts in an autocrine
fashion as a strong inducer of collagen I synthesis
[3,14] Further investigations should reveal if ET-1
eli-cits induced collagen I synthesis solely via induction of
CTGF or via additional mechanisms as it is the case
for TGF-b1 stimulation [2,55] In contrast to ET-1,
TGF-b1 induced persistent CTGF gene activation
dur-ing the stimulation period resultdur-ing in prolonged
colla-gen I synthesis, as described [41] It seems that ET-1
and TGF-b1 show different properties in regulating
the induction of collagen I synthesis by CTGF
Further examination of the intracellular signalling
mechanisms revealed an essential involvement of Gai,
PLD and PC-PLC Their inhibition abolished ET-1
induced collagen I expression The PC-phospholipases
are also important signalling intermediates for TGF-b1
induced collagen I synthesis Furthermore, it has been
reported that ET-1 and TGF-b1 require PKC-d for
induction of collagen I [30,31,70], that may be
activa-ted by DAG derived from PLD and PC-PLC Our
results demonstrate that the ET-1 and TGF-b1
signal-ling pathways terminating in induced collagen I
syn-thesis overlap partially This is in contrast to the early
postreceptor pathways, which differ between the
ET-receptors belonging to the family of G protein-cou-pled receptors and the TGF-b1 receptors as members
of the serine-threonine kinase receptor group [1,24,25] Here, we demonstrated an additive induction of col-lagen I production by ET-1 and TGF-b1 We observed
an additive CTGF gene activation by both hormones (data not shown) A further example of additive action between ET-1 and TGF-b1 is induction of myofibro-blast differentiation [71]
Some hormones acting in auto- and paracrine man-ner are capable of inducing each other’s expression [72] In human primary dermal fibroblasts, however, TGF-b1 neither induced the synthesis of ET-1 [71] nor did ET-1 induce the synthesis of TGF-b1 The inter-play between TGF-b1 and ET-1 therefore occurs within their partly common intracellular signalling pathway However, their roles may diverge because of differences in CTGF gene activation and the different effects of PLD inhibition on collagen I levels
Experimental procedures Materials
Experimental material was purchased from the following suppliers: ET-1 (human), BQ123, BQ788, TGF-b1 (human), D609, U73122, Pertussis toxin (PTX), pepsin: Calbiochem-Novabiochem, Bad Soden, Germany PD156252, neomycin trisulphate, magnesium salt of l-ascorbic acid 2- or 3-phos-phate, Ponceau Red, Coomassie blue, N-butanol: Sigma-Aldrich, Deisenhofen, Germany [125I]ET-1: Amersham⁄ Pharmacia Biotech, Freiburg, Germany Human collagen I: Sircoll, Cologne, Germany
Cell culture
Primary human dermal fibroblasts were obtained by explant culture from several biopsies of healthy skin of age-and sex-matched adult donors Fibroblasts were maintained
in Dulbecco’s modified Eagle’s medium (DMEM), supple-mented with ascorbic acid (71 lg per mL), penicillin (100 UÆmL)1), streptomycin (100 lgÆmL)1), 0.2 m glutamine (all from Biochrom, Berlin, Germany) and fetal bovine serum (10%, FBS; PAA, Linz, Austria) and cultured in the humidified atmosphere of 5% CO2 Fibroblasts were sub-cultured to confluence and used between passages 2–5
Northern hybridization analysis
Fibroblasts were seeded at 5· 104 cells per cm2 into
100 mm dishes and cultured for 24 h followed by starvation with FBS-free DMEM for 24 h Thereafter, cells were cul-tured in FBS-free DMEM in the absence or with either
Fig 7 Long-term effects of ET-receptor gene activations by ET-1.
Human primary dermal fibroblasts were cultured without or with
100 n M ET-1 for indicated times RNA was isolated and 2 lg RNA
was reverse-transcribed and subjected to Q-PCR in presence of
specific primer pairs for the ETA- and the ETB-receptor Signals
from three independent experiments were determined in triplicate
and normalized to b-actin, performed with cells from different
donors and cell isolations Unstimulated values were set as 1.0.
Results are means ± SEM Asterisks represent statistically
signifi-cant differences, P < 0.05.
Trang 9100 nm ET-1 or 180 pm TGF-b1 (daily addition) for the
indicated times Total RNA was isolated using Qiagen
RNA columns (Qiagen, Hilden, Germany) Purified RNA
(2–5 lg per lane) was electrophoresed on formaldehyde
agarose gels running in Mops buffer (40 mm Mops, 10 mm
sodium acetate, 1 mm EDTA, pH 7.0) and transferred to
nylon membranes (Amersham⁄ Pharmacia Biotech,
Frei-burg, Germany) Membranes were hybridized to an a-32
P-dCTP labelled 0.4 kbp human TGF-b1 or a 1.1 kbp human
CTFG cDNA probe using Hybrimax solution (Ambion,
Huntingdon, UK) according to the manufacturer’s
instruc-tions RNA loading and transfer was evaluated by
re-pro-bing the membranes with an 18S ribosomal RNA cDNA
probe
Real Time PCR (RT-PCR)
Two lg of purified RNA were reverse-transcribed by
reverse transcriptase core kit (Eurogentec, Cologne,
Germany) and subjected to PCR in the presence of
specific primer pairs (ETA-receptor: forward primer:
pri-mer: 5¢-TCCATTTCGTTATACACAGTTTTCTTC-3¢;
ETB-receptor: forward primer: 5¢-GCTGCACATCGTCATTGA
CAGTGATT-3¢) Primer pairs yielded products of 135 bp
for the ETA-receptor and of 150 bp for the ETB-receptor
The annealing temperature used was 60C and 40 cycles
were performed PCR products were separated using
agarose gels containing ethidium bromide and signals
visualized under UV light
For semiquantitative PCR (Q-PCR), RNA was
reverse-transcribed by qPCR core kit (Eurogentec Deutschland
GmbH, Cologne, Germany) and subjected to Q-PCR in the
presence of the specific ET-receptor subtype primer pairs in
addition to corresponding FAM-labelled probes
5¢-FAM-CAGTCCTCTGCCAGCAGCTTGTAGACA-3¢-TAMRA)
using the Taq Man 7700 Sequence Detection System (ABI
Applied Biosystems, Darmstadt, Germany) Assays were
run in triplicates and normalized to b-actin (forward
primer: 5¢-ATAGAGGTCTTTACGGATGTCAAC-3¢, FAM
CC-3¢-TAMRA)
Binding studies
Receptor binding and displacement studies were performed
as described [24] Fibroblasts were seeded (5· 104cells per
cm2) into 96 well plates and cultured for 2 days Cells were
washed 3 times with PBS at room temperature and
incu-bated with binding buffer (137 mm NaCl, 2.7 mm KCl,
6.5 mm Na2HPO4, 1.5 mm KH2PO4, pH 6.8) containing
[125I]ET-1 (25 pm) for 4 h at 4C Non-specific binding was determined in the presence of unlabelled ET-1 (200 nm)
For displacement studies, binding buffer contained either the nonsubtype specific antagonist (PD156252), the ETA-receptor selective antagonist (BQ123) or the ETB-ETA-receptor selective antagonist (BQ788) over a concentration range of 0.1 pm)1 lm After an incubation period of 4 h, cells were rinsed with PBS (4C), treated with cell lysis buffer (1% SDS, 0.1 N NaOH) for 10 min and the bound activity was determined using a c-counter
Collagen I synthesis
Fibroblasts were seeded (3· 104cells per cm2) into 35 mm dishes and cultured for 24 h in full medium, which was replaced by FBS-free DMEM (0.2 mL per cm2) with or without hormones or inhibitors as indicated, and cells were cultured for further 2 days ([73], modified protocol) Cul-ture media were collected and 1800 lL of each medium sample were incubated with pepsin (40 lgÆmL)1, at pH 2.5,
4C) overnight under rotation to cleave all proteins present
in the sample with the exception of collagen I After TCA precipitation (10% TCA, 0.1% Triton X-100), modification
of [74], samples of one experimental set were resuspended with nonreducing SDS loading buffer and applied to one gel for electrophoresis (6% polyacrylamide gel) Gels were fixed and stained with Coomassie Brilliant Blue R B-0149 (0.25%, 10% acetic acid, 25% isopropanol), followed by destaining with 10% acetic acid and 12.5% isopropanol
As an indirect loading control, cells were counted before and after stimulation to exclude that increased collagen I amounts may result from different cell numbers instead of the induction of collagen I synthesis
Immunoblot analysis
To determine ET-receptor protein expression, fibroblasts were seeded (5· 104
cells per cm2) into 100 mm dishes and cultured for 2 days Then the cells were washed three times with PBS and lysed with RIPA buffer (150 mm NaCl, 0.1% SDS, 0.5% desoxycholate, 1% Nonidet P 40, 50 mm Tris-HCl pH 8.0) to obtain total cell lysate or with membrane buffer (10 mm KCl, 1.5 mm Mg2Cl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 20 mm Hepes, pH 7.5) to obtain mem-brane fractions [75] Samples were diluted in reducing SDS loading buffer and subjected to 10% SDS⁄ PAGE Separ-ated proteins were transferred to nitrocellulose membranes (Amersham⁄ Pharmacia Biotech, Freiburg, Germany) Transfer efficiency was determined by Ponceau Red stain-ing Nitrocellulose membranes were blocked by incubation with 5% nonfat dried milk (BioRAD, Mu¨nchen, Germany)
in PBS for 1 h at room temperature Antigens were detec-ted using polyclonal antibodies diludetec-ted 1 : 200 in PBS for one h at room temperature [rabbit anti-(ETA receptor) IgG
Trang 10or rabbit anti-(ETB receptor) IgG, Chemicon, Hofheim,
Germany] The specificities of both antibodies were
demon-strated [76–78]
To determine secreted collagen I, TCA-precipitated
pepsin-resistent proteins derived from cell culture media
were resuspended in nonreducing SDS-buffer and subjected
to precast 4–12% gradient Tris-glycine polyacrylamide gel
electrophoresis (anamed Elektrophoreses GmbH,
Darms-tadt, Germany) Separated proteins were transferred and
further processed for immunoblotting as described above
Antigens were detected using antibodies diluted in PBS for
1 h at room temperature [1 : 100, rabbit anti-(collagen I)
IgG, Quartett, Berlin, Germany or 5 lgÆmL)1 mouse
anti-(collagen I) IgG, clone CP17L, Calbiochem-Novabiochem,
Bad Soden, Germany]
Primary antibodies were visualized using species-specific
secondary horseradish peroxidase-conjugated antibodies
(IgG) diluted 1 : 3000 in PBS [mouse anti-(rabbit IgG) Ig
or swine anti-(mouse IgG) Ig, Dako, Hamburg, Germany]
Resulting antigen-antibody complexes were detected by
chemiluminescence using ECL kit (Amersham⁄ Pharmacia
Biotech, Freiburg, Germany) Chemiluminescence signals
were recorded on X-ray films (Amersham⁄ Pharmacia
Bio-tech, Freiburg, Germany)
Densitometrical analysis
Gels and X-ray films were analysed by scanning
densitome-try [Imager Fluor-STMMultilmager (BioRAD, Heidelberg)]
The resulting data were evaluated using quantify one
4.0.3
Densitometric values obtained from samples of
unstimu-lated and untreated human primary dermal fibroblasts were
statistically not different from values obtained from
sam-ples of pretreated and unstimulated human primary dermal
fibroblasts However, the mean values of densitometric data
derived from unstimulated and untreated human primary
dermal fibroblasts of each experiment were set to a relative
value of ‘1’, serving as baseline value for the other data,
thereby representing the status of both groups of
unstimu-lated human primary dermal fibroblasts
Statistical analysis
Statistical analysis was performed using GraphPad Prism
version 3.02 All data were expressed as means ± SEM
and statistically analysed using an unpaired t-test
Acknowledgements
This study was supported by the Deutsche
Fors-chungsgemeinschaft (KR558⁄ 13 to TK) and by the
Koeln Fortune programme⁄ Faculty of Medicine,
Uni-versity of Cologne (to TK and CL)
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