Báo cáo khoa học: Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins Implications in mechanical stress-associated pathologies pptx

Two immediate early gene-encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine-rich protein 61 Cyr61⁄ CCN1 and connective tissue growth factor CTGF ⁄ CCN2,

Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins

Implications in mechanical stress-associated pathologies

Brahim Chaqour1and Margarete Goppelt-Struebe2

1 Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA

2 Department of Nephrology and Hypertension, University Erlangen-Nuremberg, Germany

Introduction

Basic physiological processes ranging from blood

cir-culation and the micturition reflex to the sense of

touch and cell movement are primarily initiated by

forces rather than molecules acting on cell surface

receptors and initiating cascades of biochemical reac-tions There is increasing evidence that mechanical strain plays an important role in maintaining normal tissue architecture by influencing cell function and behavior Under extremely or even moderately strained conditions (i.e., hypertension, obstruction), the cellular

Keywords

actin cytoskeleton; bladder obstruction;

fibrosis; hypertrophy; mechanical overload;

mechanotransduction; RhoA signaling; shear

stress; smooth muscle cells

Correspondence

B Chaqour, Department of Anatomy and

Cell Biology, State University of New York

Medical Center, 450 Clarkson Avenue,

Box 5, Brooklyn, NY 11203, USA

Fax: +1 718 0270 3732

Tel: +1 718 270 8285

E-mail: bchaqour@downstate.edu

M Goppelt-Struebe, Department of

Nephrology and Hypertension, University

Erlangen-Nuremberg, Loschgestrasse 8,

91054 Erlangen, Germany

Fax: +49 9131 8539202

Tel: +49 9131 8539201

E-mail: Goppelt-Struebe@rzmail.

uni-erlangen.de

(Received 10 April 2006, revised 1 June

2006, accepted 6 June 2006)

doi:10.1111/j.1742-4658.2006.05360.x

Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces These forces are signifi-cantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic over-load Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems Two immediate early gene-encoded members of the family

of the Cyr61/CTGF/Nov proteins referred to as cysteine-rich protein 61 (Cyr61⁄ CCN1) and connective tissue growth factor (CTGF ⁄ CCN2), are highly expressed in several mechanical stress-related pathologies, which result from either increased externally applied or internally generated forces

by the actin cytoskeleton Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibro-tic diseases At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated

by various components of the signaling machinery, i.e., small Rho

GTPas-es, mitogen-activated protein kinasGTPas-es, and actin binding proteins This review discusses the mechanical regulation of the Cyr61 and CTGF in var-ious tissues and cell culture models with a special attention to the cytoskel-etally based mechanisms involved in such regulation

Abbreviations

CTGF, connective tissue growth factor; Cyr61, cysteine-rich protein 61; MAP, mitogen-activated protein; SRF, serum response factor; SSRE, shear stress-responsive elements; VEGF, vascular endothelial growth factor; CCN, Cyr61/CTGF/Nov.

components of organ systems, particularly fibroblasts,

endothelial and smooth muscle cells, become subjected

to mechanical inputs beyond a normally acceptable

range This may lead to an inappropriate response of

the cells to altered types of mechanical signals The

transfer of such an excessive strain results in the

production of various growth factors, cytokines, and

hormones, ultimately leading to hypertrophic,

hyper-proliferative and⁄ or fibrotic responses For instance,

mechanical stress imposed on the vascular wall by the

intraluminal blood pressure is critical for regulating its

growth and phenotypic differentiation as shown by ex

and in vivo studies [1] Similarly, urethral obstruction

induced experimentally results in altered pattern of

stretch within the bladder wall, which triggers

hyper-trophic and fibrotic responses [2] Consistent with these

in vivo observations, in vitro studies have shown that

mechanical forces applied to and⁄ or generated by the

cells results in profound alterations of the

histo-morphometry, phenotype and function of the cells

[3–7] The onset of this process is characterized by the

activation of a cascade of signaling events coupled to

progressive and perhaps, interdependent changes of

gene expression

The cysteine rich protein 61 (Cyr61) and connective

tissue growth factor (CTGF) belong to the family of

Cyr61/CTGF/Nov (CCN) proteins, structurally related

secreted matricellular proteins with functions in

adhe-sion, migration, proliferation and extracellular matrix

synthesis [8,9] While being minimally expressed in

normally functioning quiescent adult tissues, the Cyr61

and CTGF genes are strongly up-regulated in

mechan-ically challenged organ systems from various etiologies

including hypertension, hemodynamic overload,

meta-bolic injury and obstruction These observations led to

the hypothesis that mechanical factors typified by

shear stress, tension, stretch and hydrostatic pressure

might be primary inducers of the Cyr61 and CTGF

genes in these pathological conditions [10]

Evidence in the literature indicates that the CTGF

and Cyr61 genes are rapidly induced in cultured cells

in response to physical and chemical stimuli, and that

the early expression of these genes is the precursor to

long-term modification in the cell’s phenotypical and

synthetic features [8,11] In most cases, Cyr61 and

CTGF are coinduced upon exposure of the cells to

various hormones, growth factors, inflammatory

mole-cules and apoptotic agents In particular, coinduction

of Cyr61 and CTGF occurs upon stimulation of

connective tissue type cells with transforming growth

factor-b1 (TGF-b1), vascular endothelial growth factor

(VEGF), fibroblast growth factor (FGF), angiotensin II,

prostaglandins, bioactive lipids, thrombin, factor IX,

estrogens and apoptotic agents [2,12–15] The Cyr61 and CTGF genes are also coinduced by mechanical stretch, but a higher strain level is required for induc-tion of CTGF than Cyr61 suggesting that under mechanically strained conditions their genes may not

be coordinately regulated [16,17] This notion is sup-ported by the observation that CTGF gene induction is delayed compared to that of Cyr61 in the bladder wall experiencing mechanical overload through urethral obstruction [2] Differential pattern of expression of these genes underlies their distinctively nonredundant functions despite their relatively high structural homol-ogy (40% at the amino acid level) Correspondingly, Cyr61- and CTGF-deficient mice show different phenotypes: loss of Cyr61 expression leads to early embryonic lethality due to placental insufficiency and compromised vessel integrity, while lack of the CTGF expression affects primarily the skeletal development

as a result of impaired chondrocyte proliferation and extracellular matrix production and turnover [18,19]

In this review we describe in vivo and in vitro evi-dence relating CTGF and Cyr61 to mechanical stress and discuss the molecular mechanisms of mechano-transduction leading to the induction of these multi-functional proteins The readers are referred to other reviews describing in detail the structural and biologi-cal activities of these proteins [8,9,11]

Mechanical modulation of CTGF and Cyr61 gene expression in vitro and

in vivo

Mechanical regulation in bone and cartilage Cartilage and bone provide ideal tissues for the study

of the mechanical regulation and function of Cyr61 and CTGF, because these tissues experience a wide range of strains during normal use, due to both their own cytoskeletally generated tension and external load-ing Additionally, endochondral ossification is regula-ted by many factors, including mechanical stimuli, which can suppress or accelerate chondrocyte matur-ation The role of CTGF in bone was investigated by the group of Takigawa who provided evidence that CTGF is a prohypertrophic chondrocyte-specific gene product, implicated in proliferation and differentiation

of chondrocytes, and in skeletal growth and mode-ling⁄ remodeling [20] Mechanical strain is also implica-ted in cartilage biology as either cyclic tensile strains

or shear promote cartilage growth and ossification [21] In an in vitro study, Wong et al compared the effects of tensile strain and cyclic hydrostatic pressure

on CTGF expression in primary chondrocytes [22]

Their data indicated that tensile strain induced CTGF,

whereas hydrostatic pressure was without effect,

which is in contrast to the up-regulation of CTGF in

mesangial cells exposed to hydostatic pressure [23]

Meanwhile, continuous application of mechanical

sti-mulation was also performed in vivo in experimental

tooth movement, a model for mechanical-dependent

bone growth [24] CTGF mRNA expression was

increased in osteocytes at both the compressed and the

stretched side of the teeth, indicative of complex

signa-ling pathways in both types of stress, i.e., tension and

compression

With regard to Cyr61 expression, there is evidence

showing that Cyr61 is down-regulated during

differen-tiation of mesenchymal stem cells into chondrocytes or

osteoblasts, but it is up-regulated during fracture

heal-ing, suggesting a role for Cyr61 in chondrogenesis and

bone formation [25–27] In this case, it has been

postu-lated that Cyr61 contributes to bone healing through

its angiogenic potential However, in-depth analyses of

the mechanical regulation and functional significance

of Cyr61 expression in cartilage and bone remain to be

performed given the important role of Cyr61 in

skel-etal development [28]

Tensile forces in skin disorders: role of

myofibroblasts

During wound healing, skin fibroblasts at the edge of

the wound differentiate into myofibroblasts known for

their contractile capability and their capacity to

prolif-erate and migrate generating strong contractile forces

that permit wound tissue edge closing Interestingly,

increased levels of CTGF and Cyr61 were found in

fibroblasts in closing wounds [29,30] In addition, the

specialized cases of keloids, which apparently develop

in regions of the body that are subjected to relatively

higher mechanical strain than others, are lesions highly

enriched in CTGF [31,32] The scar that persists is itself

a tissue under increased mechanical strain and contains

abnormally high levels of CTGF Thus, keloids

repre-sent another example of situation in which the

mechan-ical regulation of Cyr61 and CTGF is of relevance

TGF-b which is a major profibrotic and fibrogenic

molecule, is one of the potent inducers of CTGF gene

expression in wound healing and in various

pathophys-iological situations In scleroderma, the initial

transac-tivation of CTGF is mediated through TGF-b specific

smad signaling pathway, whereas the maintenance of

CTGF expression is independent of TGF-b signaling

[33,34] However, up-regulation of the CTGF gene is

neither always preceded nor systematically

accompan-ied by a concomitant increase of TGF-b expression In

particular, CTGF expression is increased in patients with radiation enteritis with established fibrosis with-out a concomitant up-regulation of TGF-b [35] These observations indicate that even though TGF-b is the major regulator of CTGF, additional factors must be considered to understand the physiological and patho-physiological relevance of this protein in the skin Three-dimentional collagen-1 matrices are a com-mon model system to investigate the influence of mechanical stress on the cell biology of fibroblasts [36]

In this model system, increased mechanical stress was shown to up-regulate the CTGF gene while the release

of mechanical stress led to a rapid down-regulation of CTGF expression [37,38] Similarly, CTGF was down-regulated when renal fibroblasts were cultured on top

of soft collagen matrices allowing a relaxed phenotype compared to cells cultured on rigid surfaces [39] The flexible adaptation of CTGF synthesis to differences in mechanical stress argues in favor of an important role

of CTGF in the cell’s response to both externally imposed and internally generated mechanical stress Moreover, TGF-b-mediated fibroblast differentiation was enhanced when mechanical tension was applied to cells [40] TGF-b-mediated differentiation and subse-quent matrix contraction were dependent on CTGF expression, but it was not promoted by CTGF alone [41] Fibroblast differentiation may thus be an example

of an effective cooperation between soluble mediators and environmental cues

Modulation of CTGF and Cyr61 expression

by hemodynamic forces Altered hemodynamic forces are primarily responsible for the initiation of early atherosclerotic lesions, which are located preferentially in specific regions of the arterial wall subjected to nonuniform blood flow [42,43] CTGF and Cyr61 are strongly expressed in endothelial cells of atherosclerotic lesions although a definite role for CTGF in the pathogenesis of athero-sclerotic lesions has not yet been established [44–47]

In vitro studies showed that CTGF and Cyr61 belong

to the group of genes which are strongly up-regulated

in endothelial cells exposed to nonuniform shear stress [48] Conversely, constant shear stress reduced CTGF and Cyr61 mRNA expression in primary human umbi-lical vein endothelial cells (HUVEC) [49,50] These observations are consistent with the notion that physiological shear stress protects the lining endothe-lium against fibrotic and atherosclerotic diseases which are predominantly initiated in areas of turbulent flow However, other studies reported conflicting data In particular, Eskin et al have shown CTGF mRNA

expression remained unaltered when laminar flow

was applied for 24 h in cultured HUVEC or bovine

endothelial cells, while our own data showed a

down-regulation of CTGF protein in HUVEC (Cicha and

Goppelt-Struebe, unpublished results and [51]) A

microarray analysis showed that CTGF mRNA was

up-regulated by turbulent as well as laminar flow,

which is in contrast to the in vivo situation, where

CTGF is not expressed in normal vessels exposed to

uniform laminar shear stress [52] Utilization of

differ-ent types of cells and apparatus, and various shear

stress regimens may account for the discrepancies

among data from various laboratories

Investigations of the biological effects of mechanical

forces have focused originally on endothelial cells, as

the layer of endothelial cells lining blood vessels

pro-tects the smooth muscle from the direct shearing effects

of the flowing blood However, the pulsing blood

clearly stretches the entire vascular wall including the

underlying smooth muscle layers Using an animal

model of pulmonary hypertension, Lee et al have

shown that CTGF gene expression was up-regulated in

vascular smooth muscle cells of arteries and arterioles

[53] Studies with cultured smooth muscle cells from

various tissue beds showed that Cyr61 is also strongly

but transiently up-regulated upon the application of up

to 7.5% cyclic biaxial strain to cultured monolayer

smooth muscle cells while the expression of CTGF was

unaffected at this strain level [16] The minimal strain

required to trigger CTGF up-regulation was 10% [17]

Strain-mediated up-regulation of Cyr61 in bladder

smooth muscles cells regulates the expression of several

mechano-sensitive genes including VEGF, a-actin and

avintegrin subunit genes [54] Cyr61 is also one of the

earliest genes whose expression is turned on in smooth

muscle-rich tissues (e.g., aorta and bladder) with the

onset of, and throughout the time period of,

hyperten-sion or bladder outlet obstruction [2,55]

Mechanical regulation of the CTGF gene in

kidney disorders

Altered hemodynamics have an impact on end organs

such as the kidney, and result in significant alterations

of its filtering units, the glomeruli CTGF expression

has been extensively studied in renal diseases [56–58]

Hypertension which often precipitates the development

of diabetic nephropathy in hyperglycemic individuals

was associated with increased cardiac and renal levels

of CTGF [59] High glucose and TGF-b were

identi-fied as major inducers of CTGF under these

condi-tions However, it is noteworthy that the synthesis of

renal glomerular proteins is also modulated by

mesan-gial cell stretch In particular, systemic arterial hyper-tension and conditions of impaired glomerular pressure autoregulation lead to excessive expansion and repetitive cycles of distension-contraction of the elastic glomeruli [60] An enhanced glomerular capil-lary pressure in experimental animal models was asso-ciated with an increased synthesis of extracellular matrix proteins and inflammatory mediators [61] Increased capillary plasma flow rates may add to the up-regulation of CTGF in glomeruli of diabetic rats or patients, although the increased glucose levels are con-sidered to be the major pathophysiological cause of diabetic alterations of gene expression [62,63]

Increased glomerular capillary pressure and wall ten-sion are transmitted to resident glomerular cells This process was investigated by exposing mesangial cells to cyclic stress in vitro, which transiently up-regulated CTGF [58] In another study, sustained up-regulation

of CTGF was attributed to increased hydrostatic pres-sure and was associated with the induction of program cell death of mesangial cells [23] Thus, both in vivo and in vitro studies stress an important role of mechan-ical strain in the kidney and associated pathologies However, the molecular details of mechano-transduc-tion in glomerular cells have not been investigated yet

Of particular interest is that the podocytes, forming an epithelial layer enveloping the glomerular capillaries, produce increased amounts of Cyr61 and CTGF in animal models of glomerulonephritis and diabetic nephropathy [64,65] Whether mechanical factors are the primary inducers of Cyr61 and CTGF under these conditions is unknown

How is mechanical stress translated into Cyr61 and CTGF gene expression?

Mechanistically, the transfer of excessive strain results

in the activation of multiple signaling cascades, cul-minating in the reprogramming of gene expression and the production of growth factors such as Cyr61 and CTGF Understanding the mechanisms whereby mechanical forces induce Cyr61 and CTGF gene expression is important so that mechano-transduction-based therapies and⁄ or pharmacological intervention can be formulated to prevent⁄ reverse the deleterious effects of excessive strain and mechanical overload However, the notion of separate and linear pathways linking mechanical stimuli to the expression of a mec-hano-sensitive gene is an oversimplification Instead, complex and interdependent signaling networks are probably involved

The most important sensors of mechanical stress are integrins linking extracellular matrix proteins to

intracellular signaling Organization of integrins into

focal complexes is dependent on the type of matrix

molecule and it is modulated by the physical state of

the matrix [66] Integrins are coupled via adaptor

mole-cules, such as integrin linked kinase, to the actin

cyto-skeleton and to various signaling molecules including

mitogen-activated protein (MAP) kinases and small

GTPases [67,68] The small GTPases of the Rho family

are central in mechano-transduction, mediating the

formation of focal complexes [69], and also as

trans-ducers of signals leading to changes in gene expression

and cellular shape and morphology [70] The impact of

altered cell morphology, rearrangement of focal

adhe-sion complexes and changes in F-actin structures on

CTGF expression was demonstrated when fibroblasts

were cultured in 3D collagen gels [38]

There is a clear evidence that the Cyr61 gene is

regu-lated through mechano-transduction pathways that

appear to converge at the level of cytoskeletal actin

dynamics [16] Transduction mechanisms involving

protein kinase C and phosphatidyl inositol 3-kinase

activation partly blocked stretch-induced Cyr61 gene

expression in smooth muscle cells [71] Selective

inhibi-tion of Rho⁄ actin signaling pathways altered this

stretch effect as well, and a superinduction of the

Cyr61 gene was observed upon treatment of the cells

with actin polymerization-inducing drugs alone The

Cyr61 gene appears to be particularly sensitive to the

physiological state of G-actin because the sole

treat-ment of the cells with swinholide, which induces actin

dimerization, was sufficient to induce up-regulation in

the expression of the Cyr61 gene [71] In line with these

results it was shown in NIH 3T3 fibroblasts that the

Cyr61gene belongs to a group of target genes of serum

response factor (SRF), which are dependent on

RhoA-actin signaling [72] Additionally, the promoter region

of the Cyr61 gene contains so-called shear

stress-respon-sive elements (SSRE) representing the core sequence of

NF-jB binding sites found previously in shear

stress-responsive genes in endothelial cells [73] A study by

Grote et al [74] has shown that mechanical stretch of

vascular smooth muscle cells leads to enhanced

expres-sion of the Cyr61 gene via the mechano-sensitive

tran-scription factor early growth response factor-1 (Egr-1),

a transcription factor which is up-regulated

independ-ently of cytoskeletal actin remodeling [72] Therefore,

additional studies are needed to determine the

contri-bution of both stretch-responsive and actin

dynamic-sensitive elements within the Cyr61 promoter and their

cognate transcription factors, and the relevance of these

findings in pathological conditions

While data related to the mechanical regulation of

Cyr61 are still limited, more detailed studies focused

on the regulation of CTGF In the network of interact-ing signalinteract-ing mediators, RhoA GTPase seems to play

a major role in maintaining the basal turnover of CTGF mRNA and also in the stimulated expression of CTGF Interference with RhoA signaling by toxin B

or more specifically C3 exoenzyme prevented up-regu-lation of CTGF by lysophosphatidic acid, a known activator of RhoA [75] Similarly, disruption of micro-tubuli by colchicine, which activates RhoA in a recep-tor-independent way, also activated CTGF in a toxinB-sensitive manner [76] Involvement of RhoA in CTGF expression was confirmed by overexpression

of constitutively active RhoA or dominant negative RhoA ([39] and S Muehlich & M Goppelt-Struebe, unpublished results) RhoA signaling interacts with other signaling pathways involved in CTGF expres-sion Inhibition of RhoA-associated kinase inhibited TGF-b-mediated up-regulation of CTGF, which is primarily mediated via the Smad 3⁄ 4 signaling path-way [77,78] Similarly, angiotensin-mediated induction

of CTGF requires signaling through MAP kinases and RhoA GTPase Angiotensin II-induced activation

of MAP kinase and adhesion-dependent activation of RhoA signaling converged at the level of CTGF mRNA expression renal fibroblasts [78] Furthermore, RhoA can be an important target for pharmacologi-cal interference with CTGF expression By inhibition

of the post-translational modification of RhoA, statins (hydroxymethyl glutaryl CoA reductase inhibi-tors) inhibit CTGF induction in vitro and in vivo [79– 82] The Rho-kinase inhibitors, Y27632 or fasudil, which inhibit CTGF expression in vitro, may be another way to interfere with overexpression of CTGF in vivo

Activation of RhoA increases the formation of F-actin stress fibers via downstream mediators, among them RhoA-associated kinase (ROCK) [83] Given the inhibition of CTGF expression by inhibitors of ROCK, it was obvious to investigate the direct effect

of changes in actin organization on CTGF expression

as a potential molecular mechanism of mechano-sens-ing Changes in the ratio of G- and F-actin were not only observed in experimental in vitro settings, but also detectable in vivo In diabetic glomeruli, which are exposed to increased mechanical strain, actin was found to be disorganized and the structure of the fibrillar F-actin was disrupted [84]

Recruitment of G-actin into F-actin stress fibers by jasplakinolide increased CTGF expression, whereas disruption of F-actin by latrunculin B reduced CTGF expression [76] Unexpectedly, cytochalasin D, which also rapidly disintegrated actin stress fibers, transi-ently increased CTGF [85] Both cytochalasin D and

latrunculin B enhance the cellular content of G-actin

[86], however, the availability of G-actin as

modula-tor of gene expression seems to be different upon

treatment with both agents: cytochalasin D was

shown to sequester and thus reduce the effective level

of G-actin [74,87] These data indicate that rather

than being regulated by F-actin stress fibers, the

expression of CTGF seems to be sensitive to changes

in the level of G-actin In line with this hypothesis,

overexpression of mutant G-actin that is no longer

able to polymerize into F-actin [88] significantly

reduced the expression of CTGF in endothelial cells

(unpublished result) There is increasing evidence that

G-actin plays a role as regulator of cellular traffic

and also gene transcription [89] Coactivator proteins

such as myocardin-related transcription factors bind

to G-actin as well as the transcription factor SRF

These coactivators thus connect proteins that until

recently were considered to belong to functionally

unrelated families such as transcription factors and

structural cytoskeletal proteins SRF, by interacting

with a response element located about 4 kb upstream

of the transcription start site in the CTGF promoter

seems to be involved as a link between cytoskeletal

rearrangement and CTGF transcription in endothelial

cells (Goppelt-Struebe, unpublished result)

We have recently studied the molecular mechanisms

whereby externally applied mechanical strain or stretch

regulates the expression of the CTGF gene We found

that an altered pattern of mechanical stretch in either

cultured bladder smooth muscle cells or the bladder

wall in vivo as a result of urethral obstruction induces

translocation and binding of NF-jB to a highly

con-served NF-jB binding site in the proximal promoter

region of the CTGF gene [17] Our data also indicated

that nuclear translocation of NF-jB and

transactiva-tion of the CTGF promoter can be blocked upon

disruption of actin stress fibers by a cell-penetrating

peptide containing the N-terminal sequence Ac-EEED

of smooth muscle a-actin The mechanical activation

of NF-jB appears as a consistent theme linking

mechanical stimuli to activation of various stretch- or

shear stress-sensitive genes and is associated with

destabilization of IjB, an NF-jB inhibitor The

stabil-ity of IjB in resting cells depends on its anchorage to

the actin cytoskeleton, possibly via its ankyrin repeat

domain Interestingly, stretch-dependent activation of

the CTGF promoter was also inhibited by the

RhoA-associated kinase inhibitor, Y-27632, which has been

shown both to alter the actin network and to inhibit

NF-jB binding activity by inducing cytosolic

stabiliza-tion of IjBa [90] Therefore, stretch-mediated

activa-tion of the CTGF gene promoter is coupled to

dynamic rearrangement of the actin cytoskeleton asso-ciated with IjB destabilization in bladder smooth mus-cle cells

Which biological activities do Cyr61 and CTGF manifest in mechanical stress conditions?

Mechanical stress experiments in vitro help to under-stand how internally generated and⁄ or externally imposed forces on the cells lead to changes in gene expression In these types of experiments, comparisons are made between cells cultured under static conditions and cyclically stretched or shear deformed cells How-ever, as reported for Cyr61 and CTGF, their expres-sion declined rapidly and even disappeared after a short period of mechanical deformation, when the stretching environment became the cell’s new nor-malcy The rapid reestablishment of basal expression might be indicative of an adaptive mechanism in which compensatory signaling pathways are activated to allow gene transcription to return to normal levels in the stimulated cells At these later time points, cells may more accurately represent those in vivo, which normally exist in a mechanically active environment

In pathological conditions, however, such compensa-tory mechanisms do not seem to take place because the up-regulation of Cyr61 and CTGF appeared to be both rapid and long lasting in the affected tissues [2,57] Identification of all factors that either prevent

or allow down-regulation of Cyr61 and CTGF in vivo should provide new clues on how to interfere with their uncontrolled overexpression in pathological con-ditions

However, the expression, even transient, of Cyr61 or CTGF may have long-term implications Previous studies suggested that Cyr61 can regulate the expres-sion of genes involved in angiogenesis and matrix remodeling [18,29] In agreement with this, interference with Cyr61 in mechanically stimulated cells markedly reduced mechanical strain-induced VEGF, av integrin and smooth muscle a-actin gene expression but had no effect on type I collagen, fibronectin and myosin heavy chain isoform expression [54] An intact cytoskeleton is required for Cyr61-dependent regulation of gene expression, indicating that cytoskeleton integrity is required for both Cyr61 expression and activity There-fore, Cyr61 may well be an integral part of the mechano-transduction process by promoting the expression of mechano-sensors such as integrins and⁄ or by propagating the mechanical signal to neigh-boring cells via the expression of autocrine and para-crine factors such as VEGF

CTGF is a protein which exerts its effects

character-istically by interaction with other proteins in a

syner-gistic or inhibitory manner [91,92] Furthermore,

CTGF itself is able to modulate cytoskeletal structures

[93] Regulation of CTGF by mechanical forces may

thus add to the complexity and variability of the

regu-lation of cellular communication Collectively, both

the expression and function of the immediate early

genes Cyr61 and CTGF cannot be separated from the

mechanically dynamic structures inside and outside the

cells and tissues

Conclusion

Further analysis of Cyr61 and CTGF gene activation

by mechanical forces will require obtaining more

infor-mation on the mechanical receptors involved in sensing

and converting the mechanical signals into chemical

ones (Fig 1) In particular, it is not clear whether

there are any specific regulators at the receptor level of

signal transmission, or whether targeting of Cyr61 and

CTGF is achieved by specific cofactors at the level of

cellular signaling molecules or transcription factors

The complex interactions among signaling molecules

and the actin cytoskeleton in mechanically challenged

cells probably implicate general as well as specific or

selective interactions among coactivators and

corepres-sors This needs to be addressed in future studies

Much more detailed studies are also necessary to

decipher the various levels of complexity in the

regula-tion of the Cyr61 and CTGF genes by mechanical

signals in different cell types and in various mechanical stress conditions In particular, it is critically important

to determine (i) whether or not the mechanisms involved in the mechanical regulation of the Cyr61 and CTGF genes are cell type-specific and⁄ or vary as a function of the type of mechanical stimuli, e.g., ten-sion, compresten-sion, shear deformation, etc.; (ii) whether such mechanisms operate in native cells and in the whole tissue in response to an altered pattern of mechanical signals; (iii) the extent to which mechanical signals override or cooperate with chemical signals ori-ginating from growth factors and cytokines; (iiii) the potential feed back or feed forward mechanisms, which either perpetuate the mechanical signals in pathological conditions or allow their quick resolution as demon-strated in the transient expression of both the Cyr61 and CTGF genes These types of investigations will provide the necessary information to more adequately reverse⁄ prevent the deleterious effects of Cyr61 and CTGF expression in various mechanical stress-associ-ated pathologies

Acknowledgements

This work was supported by grants from the National Institutes of Health and National Institute of Diabetes, digestive and kidney diseases R01-DK060572 and R21-DK068483 (to B.C.) and the Deutsche Forschungsg-emeinschaft SFB423-B3 (to M G.-S.)

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