© 2001 American Heart Association, Inc.
Vascular Biology |
Upregulatory Expression of Furin and Transforming Growth Factor-ß by Fluid Shear Stress in Vascular Endothelial Cells
From the Departments of Molecular Medicine (M.N., D.L., Y.S., T.I., T.T.) and Cell Biology (Y.-Q.Z., I.K.), Institute for Molecular and Cellular Regulation, Gunma University, and the Second Department of Internal Medicine (M.N., M.K.), Gunma University School of Medicine, Maebashi, Japan; the Department of Biomedical Engineering (J.A.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and the Department of Pathology (T.S., T.K., H.M.), Akita University School of Medicine, Akita, Japan.
Correspondence to Toshiyuki Takeuchi, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Showa-machi, Maebashi 371-8512, Japan. E-mail tstake{at}showa.gunma-u.ac.jp
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Abstract—Furin, a yeast Kex2-family endoprotease, converts many vasoregulatory propeptides, including pro-transforming growth factor (TGF)-ß to their mature forms. We examined whether furin expression is regulated by shear stress in vivo and in vitro. When an arteriovenous shunt was placed between the carotid artery and external jugular vein in rabbits, furin and TGF-ß were highly expressed in shear stress–loaded endothelial cells. Exposure of bovine aortic endothelial cells in culture to shear stress induced furin and TGF-ß expression in a similar manner. Molecular analysis of furin expression in bovine aortic endothelial cells revealed that shear stress increases the furin gene expression at transcriptional levels. Furthermore, TGF-ß itself increased the furin mRNA levels. Shear-mediated furin expression was partly mediated by TGF-ß because shear-induced furin mRNA levels were considerably decreased by overexpression of the truncated form of the TGF-ß type II receptor. Likewise, blockade of furin activity by a furin inhibitor significantly decreased the endothelial production of mature TGF-ß. Taken together, the results indicate that furin expression is induced and maintained by a coordination of shear stress and TGF-ß. Increased furin expression may facilitate the formation of mature TGF-ß, resulting in the enhanced effects of TGF-ß on endothelial cells and vascular smooth muscle cells in the vasculature.
Key Words: transforming growth factor-ß • furin • endoprotease • shear stress • endothelial cells
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Introduction |
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Shear stress is associated with the atheroprotective activities controlling vascular tone and maintaining the nonadhesive and nonthrombogenic properties of the endothelium.1 2 Shear stress controls the expression of a number of genes involved in the endothelial cell functions, including transforming growth factor (TGF)-ß,3 C-type natriuretic peptide,4 adrenomedullin,4 platelet-derived growth factor,5 endothelin,6 and tissue plasminogen activator.7 These vasoregulatory factors are synthesized in a precursor that undergoes cleavage at an Arg-4-X-3-X-2-Arg-1 (RXXR) site by yeast Kex2-type proprotein-processing endoproteases, such as furin and PC5 (also known as PC6).8 The motif RXXR is also found in other growth factor precursors (such as activin A), in some growth factor proreceptors (such as insulin receptor and hepatocyte growth factor receptor), in matrix metalloproteinases (MMPs, such as stromelysin-3 and membrane-type MMP1), in adhesion molecules (such as the cadherin family and integrins
3 and 6), and in many plasma proteins (including coagulation
factors VII, IX, and X and von Willebrand
factor).8 Previously, we have found that when furin substrates, such as brain natriuretic peptide and TGF-ß, are expressed, furin is also expressed in the cardiac muscle and the liver, respectively.9 10 In cardiac myocytes, furin is strongly expressed when the cells undergo hypertrophic growth by stretching.11 In the rat liver, furin and TGF-ß mRNA levels are coelevated in partially hepatectomized liver.10 Thus, it seems that furin expression is closely linked with the expression of its substrates. Human embryonal venous endothelial cells express furin and PC5. When the human embryonal venous endothelial cells reach a confluent state, PC5 expression increases, but furin expression remains unchanged.12 However, it is unclear just which enzymes are induced in physiological conditions, such as mild shear stress loading.
Endothelial cells express TGF-ß1 in a shear stress–dependent manner.3 TGF-ß is known to produce proteoglycans in bovine aortic endothelial cells (BAECs)13 and to inhibit vascular smooth muscle cell proliferation in response to vascular injury.14 15 Crawford et al16 generated mice lacking the thrombospondin (TSP)-1 gene, an activator protein of the TGF-ß precursor. The histological findings in TSP-1–null mice resemble those in TGF-ß1 gene–lacking mice. In both mice, vascular smooth muscle hyperplasia and alveolar hemorrhage were especially pronounced in the lungs, suggesting that TGF-ß regulates the proliferation of vascular smooth muscle cells and the formation of extracellular matrix.
TGF-ß is secreted in an inactive precursor composed of a
25-kDa dimer and a propeptide dimer (latency-associated peptide)
connected by the RHKR
sequence.17 The precursor is
then bound to a latent TGF-ß binding protein at the amino-terminal
side of the propeptide.18
This TGF-ß complex requires 2 processing reactions for maturation:
first, cleavage of RHKR by
furin,17 although the
latency-associated peptide and mature TGF-ß remain bound
noncovalently; second, removal of the latent TGF-ß binding protein
either by
plasmin19 20 or by
a conformational change by
TSP16 20 or
integrin
vß6.21
With the second reactions, mature TGF-ß is released from the complex.
Yet, regardless of the type of second reaction involved, furin cleavage
is always required for the first reaction.
To determine the physiological function of furin, we investigated the alteration of the furin mRNA levels and the mature TGF-ß formation mediated by furin in response to shear stress in cultured endothelial cells. Our results showed that shear stress is the potent stimulator of furin and TGF-ß expression. Molecular analysis of furin gene expression revealed the positive-feedback loop between furin and TGF-ß expression. The present study demonstrates that shear stress leads the upregulatory formation of mature TGF-ß by coordinated induction of furin and TGF-ß.
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Methods |
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Surgical Procedures
Fifteen male Japanese White rabbits weighing
3.0
kg underwent surgery to create a carotid arteriovenous shunt between
the left carotid artery and the left external jugular vein, as
described previously.22 As a
control, sham operations were performed by use of an identical
procedure without shunt formation in 3 rabbits. The shunt rabbits were
killed with a lethal injection of sodium pentobarbital at either 6, 12,
24, or 48 hours after the operation to obtain shear stress–loaded
arterial tissues. At each time point, 3 rabbits were used
to measure blood flow proximal to the arteriovenous shunt site on the
left and, at the same level, on the right control side, with the use of
an electromagnetic flowmeter. The arterial tissues were
subjected to immunostaining.
Immunostaining
The arterial tissue sections or cells
were first incubated with primary rabbit antibody to furin, PC5, or
TGF-ß1. The antibody to furin was generated in rabbits by use of a
synthetic
peptide.9 11 The
rabbit antibody to PC5 was a kind gift of Dr Nabil G. Seidah, Clinical
Research Institute of Montréal, Québec, Canada. The rabbit antibody
to TGF-ß1 was purchased from Santa Cruz Biotechnology. The secondary
antibody used was indodicarbocyanide-conjugated affinity-purified
donkey anti-rabbit IgG (Jackson ImmunoResearch).
Cell Culture and Shear Stress Procedure
BAECs isolated from the thoracic aortas were cultured
on a 0.5-mm-thick quartz cover glass and were placed on a
parallel-plate–type flow chamber (inner space size 16 mm
widex35 mm longx0.2 mm deep). The apparatus was
placed in a CO2 incubator at 37°C. The shear
stress forces were calculated on the basis of an equation described
previously.23 The flow rate
was adjusted to 15 dyne/cm2, unless stated
otherwise.
Reverse Transcription–PCR and Northern
Blot Analyses
Total RNA was extracted from BAECs and was
reverse-transcribed at 42°C for 1 hour with an
oligo(dT)17 primer. The
oligonucleotides used for polymerase chain reaction
(PCR) were 5'-GTACGGCTAC GGGCTGTTGGA-3' and
5'-TCGCCAGAGGGATCCTCGTC-3' for furin (369 bp),
5'-GGACTACTACGCCAAGGAGGTCAC-3' and 5'-GGTCAGCCACTGCCGCACAACT-3' for
TGF-ß1 (325 bp), and 5'-ATGACCACTGTCCACGCCAT-3' and 5'-GCCTGCT-
TCACCACCTTCTT-3' for GAPDH (272 bp). Northern blot analysis was
performed by using 10 µg of total RNA from BAECs by probing with the
mouse furin cDNA (924 bp), as described
before.9 10 11
Luciferase Assay
We used the TATA box–containing furin promoter as
described before.24 Three
5'-upstream DNA fragments, -3633/+55
(PstI-PstI),
-612/+55
(XbaI-PstI),
and -56/+55
(BamHI-PstI),
were placed before a firefly luciferase gene supplied with the
Luciferase Reporter Assay System (Promega). Each of the 3 luciferase
gene constructs was transfected to BAECs by using a TransFast liposomal
transfection reagent (Promega). Twenty-four hours after the
transfection, the medium was changed, and the culture was continued
another 6 hours with or without shear stress. Then, the cells were
harvested to prepare cell lysates for luciferase assay. Luciferase
activity is expressed as multiplicity (fold value) against the value
obtained by the (-56/+55) luciferase gene under shear
stress.
Assessment of Mature TGF-ß Formation
Mature TGF-ß formation was assessed by 3 methods:
immunoblotting, ELISA, and cell
proliferation–inhibiting activity. For immunoblotting,
the BAECs were cultured for 12 hours with or without a shear stress of
15 dyne/cm2. The medium (40 mL) conditioned
by the culture was immunoprecipitated with mouse anti–TGF-ß antibody
(R & D Systems) at 4°C for 3 hours and then with protein G–Sepharose
4FF (Amersham Pharmacia) for 1.5 hours. Proteins eluted from the
Sepharose were separated by a 10% SDS-PAGE under a nonreducing
condition and then blotted onto a nitrocellulose membrane for probing
with rabbit anti-human TGF-ß1 antiserum (Santa Cruz Biotech) at a
dilution of 1:350.
To suppress proteolytic activity in each cell lysate, we used the furin inhibitor decanoyl-arginyl-valyl-lysyl-arginyl-chloromethylketone (decRVKR-CMK, a kind gift of Dr W. Garten, Philips University, Marburg, Germany). The inhibitor was used at a final concentration of 25 µmol/L, which did not affect the cell viability up to 12 hours.11 The potency of the inhibitor was assessed by pyr-Arg-Thr-Lys-Arg-methylcoumarylamide (Peptide Institute) as a substrate.11
ELISA was performed by using a Quantikine Human TGF-ß1 Assay Kit (R & D Systems) in which a soluble TGF-ß type II receptor–precoated multiwell plate was used to absorb the TGF-ß1.
TGF-ß bioactivity was measured by a growth inhibition assay. Mink lung Mv1Lu cells (CCL64, American Tissue Culture Collection) were plated in 24-well plates at a density of 2x104 cells per well in 0.5 mL DMEM with 10% FBS. Sixteen hours after the addition of the conditioned medium, cells were incubated with 1.0 µCi/mL [3H]thymidine for 2 hours, and the radioactive counts incorporated into DNA were then determined.
Construction of an Adenovirus Vector for
TGFßRII
The truncated human type II TGF-ß receptor
(TGFßRII) was generated by using PCR with the sense primer
5'-TCGGTCTATGACGAGCAGCGG-3' and the antisense primer
5'-AGCGACCTTTCCCCACCAGG-3'.25
The TGFßRII cDNA was subcloned into a pCEP4 vector (InVitrogen),
and the expression unit was transferred to the cassette cosmid pAdex
vector.25 This cosmid
pAd-TGFßRII was subjected to a homologous recombination with the
EcoT22I-digested DNA-terminal
protein complex of Ad5-dlX in human embryonal kidney 293 cells for
generating a recombinant virus Ad-TGFßRII. A virus titer was
determined by plaque-forming units with the use of 293 cells and
expressed as a multiplicity of infection (MOI). Because the adenoviral
expression of ß-galactosidase was observed for 70% of BAECs by MOI
20 and for 95% of BAECs by MOI 50, we performed Ad-TGFßRII
infection by MOI 50.
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Coexpression of Furin and TGF-ß After Arteriovenous Shunt in Rabbit Endothelial Cells
TGF-ß is reportedly processed by furin in furin-deficient LoVo cells by infecting furin-expressing and pro-TGF-ß–expressing vaccinia recombinants.17 We initially examined the coexpression of furin and TGF-ß in arteriovenous shunt–generated rabbits. Although furin did not virtually stain in the endothelial cells of the control carotid artery, shear stress, induced via an arteriovenous shunt, caused a marked increase in staining with time, up to the 24-hour point (Figure 1
, left). The expression of furin was more marked in
endothelial cells compared with cells in the
surrounding smooth muscle.
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TGF-ß also stained increasingly up to 24 hours after the
operation
(Figure 1
, right), and as with furin expression, the staining
was more marked in endothelial cells compared with
cells in the smooth muscle. Thus, the coordinated increase of furin and
TGF-ß may suggest that pro-TGF-ß is processed by
furin.
Coexpression of Furin and TGF-ß by Shear
Stress in BAECs
BAECs were subjected to a shear stress of 15
dyne/cm2 for up to 24 hours. With shear
stress, furin and TGF-ß messages similarly increased in PCR-amplified
band intensity from the 6-hour to 24-hour points
(Figure 2A). To examine a quantitative increase by PCR
amplification, Northern blot was performed for a 6-hour point. The
increase in furin mRNA by PCR and by Northern blot was similar (both an
2.3-fold increase,
Figure 2B). Thus, coelevation of furin and TGF-ß by shear
stress was also confirmed in BAECs.
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We next examined shear stress–dependent expression of furin
and PC5 by immunostaining. Both furin and PC5 are
Kex2-family endoproteases and have similar domain structures. But only
furin has a transmembrane domain, which makes it a resident protein of
trans-Golgi networks, whereas PC5 is a cytosol
protein.8 Furin was expressed
only a little in the static condition, but the expression increased
with shear stress at 15 and 60 dyne/cm2
(Figure 2C, a through c). Furin
immunostaining was localized adjacent to the nuclei,
which is a typical feature of Golgi-resident proteins. In contrast, PC5
was distributed over the entire cytoplasm, and its staining intensity
was similar up to 60 dyne/cm2
(Figure 2C, d through f). Thus, furin expression is shear
stress dependent, whereas PC5 is stress
independent.
Transcriptional Control of Furin Expression by
Shear Stress
To determine the shear stress–mediated induction of
furin expression at transcriptional levels, we examined the shear
stress effect in the presence of actinomycin D, a potent
inhibitor of gene transcription. Shear-induced furin mRNA
increase is significantly blunted by actinomycin D, suggesting the
involvement of the transcriptional control of furin expression
(Figure 3A). We next examined the de novo protein synthesis
for shear stress–induced furin expression. To this end, we treated the
BAECs with a protein synthesis inhibitor, cycloheximide,
before their exposure to shear stress. The furin mRNA levels were
increased by shear stress at the 6-hour time point even in the presence
of cycloheximide, whereas no measurable induction was observed at the
12-hour time point, suggesting the involvement of de novo protein
synthesis for the late induction but not for the early induction of the
furin mRNA levels. In the early phase, preexisting transcription
factors are presumably activated by a shear stress–mediated
signaling pathway, resulting in the activation of the furin promoter.
However, in the late phase, these preexisting factors are spent out,
and de novo factors are not replenished by cycloheximide, resulting in
the loss of furin promoter activation despite shear stress
stimulation.
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Activation of the Furin Promoter by Shear
Stress
To further confirm the transcriptional control of the
furin gene, we performed transient transfection assays by using the
reporter gene containing the furin promoter spanning from -3633 to
+55 in front of the luciferase gene, which is referred to as
(-3633/+55)Luc.24 Exposure
of the transfected cells to shear stress for 6 hours increased the
luciferase activity of the construct (-3633/+55)Luc by 2-fold
compared with the static condition, indicating that the furin promoter
is responsive to shear stress
(Figure 3B). This increase in luciferase activity was not
artificial because transfection of BAECs with the promoterless
luciferase construct did not yield any luciferase activity by shear
stress. To narrow down the shear stress–responsive region, the
constructs containing the shorter promoter were transfected into BAECs.
(-612/+55)Luc exhibited a decrease in the shear stress–mediated
induction of the promoter activity as well as in the basal promoter
activity. Deletion to the position at -56 further attenuated the
response to shear stress. These results suggest that shear
stress–induced activity of the furin promoter is mediated through the
region between -3633 and -612 as well as between -612 and
-56.
TGF-ß Induces the Expression of Furin
TGF-ß is known to induce the expression of its own
gene and the expression of
furin.26 27 In
BAECs, both messages increased dose-dependently with
10-10 and
10-9 mol/L
TGF-ß during a 6-hour treatment
(Figure 4A). We hypothesized that TGF-ß induced by shear
stress mediates the upregulation of furin and TGF-ß. To examine the
interactions between shear stress and TGF-ß effects on furin
expression, we used TGFßRII-expressing endothelial
cells to block the TGF-ß signal pathway. After TGFßRII
expression in BAECs, the shear stress effect on the expression of furin
was decreased compared with the effect on the expression in untreated
cells
(Figure 4B versus
Figure 2A). However, the elevation of furin expression was
constantly observed 6 hours after shear stress even by expressing
TGFßRII with a high MOI number up to 100. Thus, the expression of
furin appears to occur partly through a shear stress–specific signal
pathway distinct from a TGF-ß signal pathway.
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Processing of TGF-ß Precursor by
Furin
After exposure to a static or shear stress–loaded
culture for 12 hours, we examined the formation of a 25-kDa mature
TGF-ß dimer by PAGE under a nondenaturing condition. The high
molecular weight band of 175 kDa appears to be a precursor complex of
TGF-ß with latent TGF-ß binding protein. The 25-kDa TGF-ß
increased after shear stress
(Figure 5A), as reported
previously.28 However, the
25-kDa form decreased toward the control level in the presence of the
furin inhibitor decRVKR-CMK, suggesting that furin is
involved in processing of the TGF-ß precursor.
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In a 12-hour static or shear stress–loaded culture, the
media were activated with 0.1 mol/L HCl, and the protein levels
of TGF-ß were obtained by ELISA. In the static culture, the TGF-ß1
secreted in the medium measured only 100 pg per
106 cells per 12-hour culture
(Figure 5B). In contrast, shear stress increased the
receptor-bound TGF-ß1 level to nearly 10 ng per
106 cells per 12-hour culture. However, the
TGF-ß1 level decreased to 1.5 ng per 106
cells per 12-hour culture in the presence of the furin
inhibitor decRVKR-CMK, representing
approximately one seventh of the level observed in the absence of the
inhibitor. Thus, shear stress significantly increased the
production of a mature form of TGF-ß.
Shear stress increased the inhibitory effect of
TGF-ß on the incorporation of
[3H]thymidine in Mv1Lu cells by 38%
(Figure 5C). However, this effect was reversed to control
levels in the presence of the furin inhibitor decRVKR-CMK.
Thus, the production of bioactive TGF-ß is enhanced by furin
in BAECs.
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The shear stress–loaded culture system has been used to identify a number of vasoregulatory factors involved in the physiological functions of vascular endothelial cells.1 TGF-ß1 is a factor that is elevated by shear stress.3 6 28 Exposure of BAECs to increased shear stress induces TGF-ß1 via a signal transduction pathway modulated by K+ channel currents, and this induction is regulated at the transcription level.3 To generate a bioactive TGF-ß1, the first processing reaction takes place by furin in the trans-Golgi network.17 We demonstrated the coordinated expression of TGF-ß1 and furin in shear stress–loaded BAECs and endothelial cells of rabbits with a carotid arteriovenous shunt. Interestingly, TGF-ß1 itself and furin are also induced in static culture on stimulation with TGF-ß1.26 27 Furthermore, the furin inhibitor decRVKR-CMK extensively reduced the production of the mature form of TGF-ß1 induced by shear stress. These results suggest that furin is a genuine processing enzyme for pro-TGF-ß. Interestingly, in mouse embryos at early stages, a striking degree of overlap is noted in the distribution of the TGF-ß1 family proteins and furin.29 Both proteins are highly expressed in extraembryonic mesoderm, in which vascular endothelial growth factor receptor Flk-1 is present and endothelial precursor cells are derived. Thus, the coexpression of furin and TGF-ß1 is tightly coupled.
In the present study, we provide evidence that furin expression is induced by shear stress at the transcriptional level. A number of endothelial genes are reportedly regulated by shear stress.1 2 Resnick and Gimbrone6 have identified the shear stress response element (SSRE), 5'-GAGACC-3', within the platelet-derived growth factor-B chain promoter and have shown that SSRE is present in many shear stress–inducible genes. Our 5'-deletion analysis suggests that a proximal promoter region up to -56 is not sufficient to be responsive to shear stress and that the region between -612 and -56 is necessary for shear stress response. Furthermore, the region upstream from -612 contributes to the increase in furin promoter activity by shear stress. Searching for the SSRE within the promoter region between -3633 and +55 revealed that SSREs are located at -419, -817, -2337, -3199, and -3626 (accession X15723), suggesting that these multiple SSREs are responsible for the shear stress–mediated furin gene expression.
Induction of the furin expression by TGF-ß1 merits further
discussion. Overexpression of the TGFßRII did not abolish the
early increase in furin mRNA levels by shear stress, but it did inhibit
its later increase. These results suggest that the late increase is
induced by TGF-ß1. Because furin produces mature TGF-ß1, the
positive-feedback loop, which links shear stress–induced furin and
TGF-ß expression to a further generation of mature TGF-ß by
TGF-ß–induced furin, may enhance the effect of TGF-ß on
endothelial cells and vascular smooth muscle cells.
When TGF-ß1 is overproduced in rat arterial
endothelium by an adenovirus vector, transduced
endothelium developed a cellular and matrix-rich
neointima with cartilaginous metaplasia of the vascular
media.30
It is well appreciated that TGF-ß plays pleiotropic effects on vascular cells. TGF-ß strongly accelerates lesion formation by increasing cellularity and markedly inducing extracellular matrix fibrosis. An increase in active TGF-ß levels is associated with the progression of vascular lesions. A soluble form of TGFßRII was effective in preventing negative remodeling with adventitial fibrosis and neointima formation in an arterial balloon injury in rats.31 To block the negative effect of TGF-ß, modification of TGF-ß synthesis by a furin inhibitor may be therapeutically useful in the treatment of atherosclerotic vascular lesions. In contrast, in the lesion-free endothelium, TGF-ß provides an atheroprotective function by stimulating proteoglycan formation13 and inhibiting vascular smooth muscle cell proliferation.14 Physiological levels of TGF-ß1 appear to suppress cell division partly by inhibiting the expression of vascular endothelial growth factor receptors, such as Flk-1, in endothelial cells.32 We suggest that the coordinated upregulatory loops between furin and TGF-ß expression in the shear stress–loaded vasculature favor an atheroprotective role of TGF-ß in lesion-free vascular endothelial functions.
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Acknowledgments |
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This work is supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture. We thank Eiko Hamana for her secretarial assistance.
Received October 10, 2000; accepted January 22, 2001.
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