Arch Dermatol Res DOI 10.1007/s00403-014-1514-2
CONCISE COMMUNICATION
Cyclic mechanical strain induces TGFb1-signalling in dermal fibroblasts embedded in a 3D collagen lattice Andreas S. Peters • Georg Brunner Thomas Krieg • Beate Eckes
•
Received: 7 May 2014 / Revised: 30 September 2014 / Accepted: 7 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Many tissues are constantly exposed to mechanical stress, e.g. shear stress in vascular endothelium, compression forces in cartilage or tensile strain in the skin. Dermal fibroblasts can differentiate into contractile myofibroblasts in a process requiring the presence of TGFb1 in addition to mechanical load. We aimed at investigating the effect of cyclic mechanical strain on dermal fibroblasts grown in a three-dimensional environment. Therefore, murine dermal fibroblasts were cultured in collagen gels and subjected to cyclic tension at a frequency of 0.1 Hz (6 cycles/min) with a maximal increase in surface area of 10 % for 24 h. This treatment resulted in a significant increase in active TGFb1 levels, leaving the amount of total TGFb1 unaffected. TGFb1 activation led to pSMAD2-mediated transcriptional elevation of downstream mediators, such as CTGF, and an auto-induction of TGFb1, respectively. Keywords Mechanoregulation Tension Skin fibroblast CTGF
A. S. Peters (&) Department of Vascular and Endovascular Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany e-mail: [email protected] G. Brunner Department of Cancer Research, Fachklinik Hornheide, Mu¨nster, Germany T. Krieg B. Eckes Department of Dermatology, University of Cologne, Cologne, Germany
Introduction Cells and their surrounding tissues are constantly exposed to mechanical stress; many cellular functions rely on the existence of stress, some are even dependent on it, exemplified clearly by the action of gravity onto the musculoskeletal system since its absence causes bone and muscle atrophy in astronauts in space [36]. Other examples for the action of mechanical stress are compression forces in cartilage, shear stress in the vasculature, contraction forces in muscles and tendons as well as interstitial fluid pressure in tumour stroma [10, 29, 32]. They all influence cellular signalling and the properties of the respective tissues. Enhanced deposition and cross-linking of extracellular matrix (ECM) components surrounding a tumour is caused by ECM stiffness and is also involved in the development of malignancy and metastasis [24]. Enhanced stiffening also contributes to scarring and fibrosis and results in less pliable connective tissue. Alterations in stiffness are sensed by resident fibroblasts that display an activated phenotype and maintain the fibrotic response [12, 39]. With increasing age fibroblasts interact with a defective ECM leading to changes in skin homeostasis with altered production of ECM and proteases [11]. The conversion of external mechanical stimuli into intracellular biochemical signalling can be accomplished in various ways: the best characterised one is via integrins which cluster in focal adhesions and connect the ECM with intracellular actin microfilaments [10, 33]. Other transduction pathways include mechanosensitive ion-channels or cilia [35, 40]. Mechanically regulated genes have been investigated in various cell types and tissues, which are exposed to mechanical stress, like bone, endothelium and smooth muscle [17]. The extent of mechanical force in terms of
123
Arch Dermatol Res
tensile stress in healthy skin varies with anatomical location and can amount to several kPa. In fibrotic or desmoplastic tissue, forces can exceed 3 gPa [8]. Cultured fibroblasts can experience forces of \50 Pa if seeded into freely contracting collagen gels and up to [1 gPa if plated in monolayer cultures (ML) [14, 23]. Collagen I (COL I) is the most abundant connective tissue constituent in the skin. Hence, characterization of cells, cultured in a three-dimensional collagen matrix, is a well-established method to mimic the in vivo situation [3]. If attached to the perimeter of the well, the collagen lattice is prevented from contraction leading to a self-generated tensional force acting on the cells. Under these conditions human skin fibroblasts differentiate into a-smooth muscle actin (aSMA) positive myofibroblasts showing a bipolar morphology and are characterized by increased ECM synthesis and production of transforming growth factor b (TGFb) and connective tissue growth factor (CTGF), among other profibrotic factors [7, 21, 37, 38]. Hence, this culture model reflects conditions similar to those which are met in fibrotic and desmoplastic tissues [2, 16]. A key protein secreted by myofibroblasts and other mesenchymal cells in fibrosis is CTGF [22]. It has been demonstrated that the gene expression of CTGF is increased in fibrotic skin disorders [19]. Mechanical stress affects CTGF expression in a cell type- and stress-specific way: tension exerted on chondrocytes and bone causes an increase in CTGF, while fluid shear stress in endothelial cells leads to a reduction [9, 43, 44]. CTGF is most potently induced by TGFb1 and endothelin-1 (ET-1), although independent regulation has also been reported [18]. Production of active TGFb1 follows a complex pathway. First, the latent, inactive pro-form is produced consisting of the growth factor itself covalently attached to the latency associated peptide (LAP). Secretion of the proform is facilitated by binding of the latent LAP–TGFb1 complex to the latent TGFb-binding protein 1 (LTBP-1) which anchors the entire latent complex to the ECM via fibrillin and fibronectin [26, 31]. Activation of TGFb1 is then accomplished in the extracellular space either by proteolysis or via av integrins [25, 26, 34]. Our previous work showed that the application of cyclic mechanical stress to primary murine fibroblasts seeded as ML resulted in decreased CTGF and aSMA levels by a mechanism that is independent of TGFb1 but downstream of ET-1 [28]. This work provided a mechanistic explanation at the molecular level for the previously reported downregulation of CTGF and aSMA in human skin and lung fibroblasts [4, 20]. This study aimed at investigating the adaptive response of dermal fibroblasts to cyclic mechanical strain, focussing on production and activity of the profibrotic factor TGFb1
123
and corresponding downstream signalling. Fibroblasts were seeded in collagen lattices providing the cells with a 3D surrounding, similar to the in vivo situation—a culture model reflecting conditions which are met in fibrotic diseases.
Methods Culture of mouse dermal fibroblasts Primary murine dermal fibroblasts were isolated from the skin of newborn C57BL6 mice according to standard procedures and cultured in DMEM supplemented with 10 % foetal calf serum (FCS) as described [28]. Fibroblasts were used in passage 2–5. All animal protocols were approved by the local veterinary authorities (LANUV NRW 8.87-50.10.31.8.197). Application of cyclic strain All experiments were carried out using an FX-4000T Flexercell Tension Plus (Flexcell International Corporation, Hillsborough, NC, USA). Cells were seeded at a number of 0.5 9 106 in a total volume of 2 ml containing a final concentration of 0.3 mg/ml collagen I (Curacyte, Leipzig, Germany) as described [21]. The cell suspension was transferred to 6-well Tissue Train Plates (Flexcell International Corporation), each well having a flexible silicon membrane at the bottom and a circular foam ring attached to the perimeter of the well. The plates were incubated at 37 °C for 1 h to allow the collagen to polymerize. When using serum-free conditions, cells were washed thoroughly three times with PBS before mixing with the collagen solution, which was prepared free of FCS. Cyclic mechanical tension was applied in a sinusoidal pattern (0.1 Hz, 24 h) with a maximal equi-biaxial increase in surface area of 10 % (similar to [28, 32] ). Cells cultured with the identical collagen suspension in the same plates, but not exposed to cyclic mechanical strain, were used as controls. Isolation of RNA and RT-PCR Collagen gels were washed with PBS, transferred to a 2 ml Eppendorf tube containing 300 ll of RLT buffer (Qiagen, Hilden, Germany) and a 5-mm steel bead and homogenised using a Mixer Mill MM 300 (Retsch, Haan, Germany) at a frequency of 30 Hz for two minutes. Total RNA was isolated using the RNeasy Mini-Kit (Qiagen). Reverse transcription and semi-quantitative RT-PCR were performed as described [28] using the following primers: TGFb1 forward caa-cgc-cat-cta-tgagaa-aac-c, TGFb1 reverse aag-
Arch Dermatol Res
ccc-tgt-att-ccg-tct-cc (300 bp); CTGF forward cac-cta-aaatcg-cca-agcc, CTGF reverse gcc-atg-tctccg-tac-atc-ttc (271 bp); S26 forward aat-gtg-cag-ccc-att-cgc-tg, S26 reverse cttccg-tcc-tta-caa-aac-gg (324 bp). Intensity of bands corresponding to the analysed transcripts was quantified by densitometry using Image J (http://rsb.info. nih.gov/ij) and normalised to the intensity of S26 signals. Protein isolation and western blotting Collagen gels were washed with PBS and directly lysed by ultrasound in RIPA buffer [150 mM NaCl, 50 mM Tris– HCl, 1 % NP-40, 0.1 % SDS, 0.5 % Na-deoxycholate, 5 mM EDTA, 1 % Triton X-100 (Sigma-Aldrich, Steinheim, Germany), pH 8] containing protease- and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by SDS-PAGE, blotted and incubated with antibodies directed against pSMAD2 (Ser465 and Ser 467; Cell Signalling Technology, Frankfurt, Germany) in 5 % non-fat milk in TBS-T pH 7.6, followed by incubation with secondary HRP-conjugated antibodies (Dako, Glostrup, Denmark). Signals were detected using ECL (Thermo Scientific, Langenselbold, Germany). Membranes stained
Fig. 1 Cell morphology and gene regulation of murine dermal fibroblasts embedded in a 3D collagen lattice. Fibroblasts were seeded in collagen gels supplemented with 10 % FCS and exposed to cyclic mechanical tension (strained) or not (tethered) for 24 h. Cyclic stress created an increase in surface area of 10 % at a frequency of 0.1 Hz (96/min). Fibroblasts in both culture conditions showed actin stress fibres (F-actin, red; DAPI depicts nuclei, blue; magnification 9100).
with Ponceau S prior to antibody incubation were used as loading control. Determination of TGFb bioactivity TGFb1 levels were assessed in serum-free media supplemented with 0.1 % BSA (Low Endotoxin; PAA, Duesseldorf, Germany) that was conditioned for 24 h by fibroblasts. TGFb1 activity was determined using mink lung epithelial cells stably transfected with a luciferase reporter under the control of a truncated PAI-1 promoter (kind gift by Dr. Daniel B. Rifkin, New York University Medical Center) as described [1]. Immunohistochemistry Collagen gels were fixed in 4 % paraformaldehyde (PFA) in PBS for 15 min and processed as described [21] to visualise filamentous (F-) actin with Alexa Fluor 594 phalloidin (1:100 in 1 % BSA; Invitrogen, Eugene, OR, USA). Nuclei were counterstained with DAPI. Gels were transferred onto glass slides and mounted (Immu-Mount, Thermo Scientific). Photomicrographs were acquired using
a Fibroblast showed parallel alignment when strained or b were randomly oriented when left at rest. c Transcript levels for TGFb1 and CTGF were assessed by semi-quantitative RT-PCR. S26 RNA was used for normalisation. Bar graph shows quantitative analysis of signal intensity. Intensities of strained cultures (black) are displayed relative to tethered ones (grey) which were set at 1 (n = 3)
123
Arch Dermatol Res
an Olympus IX 71 microscope equipped with a DeltaVision system (Applied Precision, Issaquah, WA, USA). Viability assay Collagen gels were produced as described containing 0.5 9 106 cells and cultured for 24 h in DMEM supplemented with 10, 1 or 0 % FCS. Gels were washed thoroughly three times with PBS, stained with 0.4 % trypan blue solution (Sigma-Aldrich) and washed again three times with PBS. Living as well as dead cells were counted using a light microscope in four non-overlapping fields at 2009 magnification. Statistical analysis Statistical analysis was carried out using unpaired Student’s t test (in case of comparison of more than two groups prior analysis of variance was performed). Results are presented as mean ± standard deviation. A p value \0.05 was considered to be significant.
Results To assess the adaptive response of skin fibroblasts to cyclic mechanical tension, primary murine dermal fibroblasts
Fig. 2 TGFb1 bioavailability and -signalling in strained and tethered cultures. Fibroblasts were seeded in collagen gels and either exposed or not to cyclic stress for 24 h under serum-free conditions. TGFb1 bioactivity was determined in culture supernatants either directly or following heat activation reflecting the amount of active or total TGFb1, respectively. a Active TGFb1 is increased under strained (black, grey = tethered) conditions (**p = 0.00171) while total TGFb1 is unaltered. b The fraction of activated TGFb1 was even
123
were cultured in three-dimensional collagen lattices in complete medium and either exposed to equi-biaxial strain in a sinusoidal manner at 0.1 Hz (6/min) to attain an increase in surface area of 10 % (strained) or left at rest (tethered) for 24 h. Strained fibroblasts showed a parallel alignment (Fig. 1a) while tethered ones were randomly dispersed (Fig. 1b). In both conditions, fibroblasts assumed an extended bipolar shape and expressed pronounced stress fibres indicated by the staining (red) for F-actin (Fig. 1a, b). Application of cyclic mechanical tension induced a two or threefold increase in TGFb1 and CTGF transcript levels, respectively, as indicated by semi-quantitative RT-PCR (Fig. 1c). To determine whether the increased transcription levels resulted in elevated levels of secreted-bioactive TGFb1, serum-free collagen gels were subjected to cyclic mechanical strain for 24 h or left unstrained. The conditioned media were tested for the amount of total and active TGFb1 produced using the mink lung epithelial cell bioassay as described [1, 5, 28]. Supernatants of strained cultures showed a significant increase (**p = 0.00171) in active TGFb1 by 44 % compared to tethered controls while the amount of total activatable TGFb1 was unaffected (Fig. 2a). Consequently, the fraction of activated TGFb1 showed a further enhanced twofold increase with respect to strained fibroblasts (***p = 7.9331 9 10-5, Fig. 2b). To
further enhanced for strained (black, grey = tethered) cultures (***p = 7.9331 9 10-5). TGFb1 signalling was analysed in cell lysates by western blotting by detecting pSMAD2. Ponceau S staining of membranes after blotting indicates equal loading. c Elevated pSMAD2 signals were detected in strained compared to tethered cultures. Intensities of strained cultures (black) are displayed relative to tethered ones (grey) which were set at 1 (n = 3)
Arch Dermatol Res
many roundly rather than bipolar-shaped cells (Fig. 3b, c). However, in principle parallel orientation of fibroblasts in strained lattices was preserved (Fig. 3b).
Discussion
Fig. 3 Cell viability. a Fibroblasts were seeded in collagen gels with three different concentrations of FCS (10, 1 and 0 %) for 24 h. Collagen gels were incubated with 0.4 % trypan blue solution staining dead cells. Live as well as dead cells were counted using a light microscope in four non-overlapping fields at 9200 magnification. Results are presented as percentage live of total cells. Decreased FCS levels correlated with significant reduction of living fibroblasts (10 vs. 1 %: ***p 4.1943 9 10-6, 10 vs. 0 %: ***p 4.1912 9 10-5, 1 vs. 0 %: *p 0.0168; prior to analysis by t test, overall difference of means between the three groups was shown to be statistically significant by analysis of variance: ***p = 4.2109 9 10-7). b, c Fibroblasts were seeded in collagen gels and either exposed or not to cyclic stress for 24 h under serum-free conditions. Among reduced cell viability many cells appear rather roundly than bipolar shaped. However, the principle of parallel alignment of fibroblasts in strained cultures is preserved (F-actin, red; DAPI depicts nuclei, blue; magnification 9100)
assess whether the difference in bioactive TGFb1 is reflected by downstream signalling of TGFb1, pSMAD2 levels were determined in strained and resting cultures at the protein level by western immunoblotting. Levels of pSMAD2 were fourfold elevated in strained vs. tethered cultures (Fig. 2c), clearly confirming differential regulation of TGFb1 bioactivity by cyclic mechanical tension. To assess the effects of serum-free conditions in the 3Dculture system on primary murine skin fibroblasts, a viability assay was carried out using trypan blue solution to identify dead cells at three different concentrations of FCS (10, 1 and 0 %). Comparison of the three FCS concentrations revealed a highly statistically significant reduction in the amount of live cells with decreasing FCS concentration (Fig. 3a). Staining flexed as well as tethered cultures for F-actin (red) confirmed reduced cell viability, also showing
In this study we show that cyclic (equi-biaxial) mechanical tension induced TGFb1 signalling as well as increased expression of CTGF in primary murine skin fibroblasts. This result appears to contradict our previous report showing that myofibroblast differentiation was attenuated upon the action of tensile strain in ML [28]. However, ML cultures are devoid of bioactive TGFb1, whereas in 3D collagen lattices—both in strained as well as tethered— active TGFb1 was detected at 100–200 pg/ml, clearly exceeding the threshold at which fibroblasts are responsive. TGFb bioactivity was confirmed by enhanced phosphorylation of SMAD2. Under strained conditions amounts of total TGFb1 protein were unaltered compared to tethered controls. We were unable to assess whether TGFb1 activation is independent of protease activity; however, based upon prior studies demonstrating a stretch-induced conformation of LAP, that is independent of protease activity, we propose, that stretching of the collagen lattice may result in a protease-independent, stretch-induced activation of TGFb1 [42]. It has been shown before in rat mesangial cells cultured in monolayer and subjected to cyclic mechanical tension that TGFb1 was increased at both transcriptional as well as protein level (active and latent); interestingly in that study the fraction of active TGFb1 in stressed cultures remained unchanged compared to static controls [30]. Of note, regulation of latent TGFb activation appears to be cell- and stress-type specific, since uni-axially applied cyclic tension was reported to cause decreased SMAD-signalling in rat cardiac fibroblasts grown in a 3Dcollagen matrix [13]. Conducting experiments under serum-free conditions with the aim to exclude the interference of serum-derived growth factors and to reveal the bare effect of cyclic mechanical tension was difficult because of severely reduced cell viability. Although the culture of skin fibroblasts in collagen gels is closer to the in vivo situation by providing a 3D environment, which can be modulated by the application of force, the system falls short of several important features: Although collagen I is the most abundant ECM structural protein in skin, the 3D collagen lattice remains a single-component matrix offering adhesive properties and structural integrity but lacking other important components, e.g. elastin [27]. Also, in comparison to human dermis, the protein density is lower by a factor of approximately 103, while the water content with about 99 % is immensely high [6, 27]. In the 3D lattice,
123
Arch Dermatol Res
collagen monomers are laterally cross-linked to form fibres, which are merely entangled instead of covalently bound [41]. This can lead to so called non-affine deformation: the strain is not evenly distributed throughout the lattice due to fibre slippage, resulting in uniform dissipation of stress [27]. By contrast, ML does not deform in a nonaffine way, but rather deforms as a continuum [15]. To conclude, we show here for the first time that dermal fibroblasts grown in a collagen gel, providing them with a 3D surrounding mimicking the in vivo situation, and subjected to cyclic mechanical tension show elevated levels of bioactive TGFb1 and corresponding downstream signalling. By contrast, ML cultures provide an excellent tool to study effects based solely on biomechanical tension acting on fibroblasts without confounding influences of biologically active TGFb. Acknowledgments We thank Gabriele Scherr for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft through SFB 829 (to BE and TK) and KR 558/14.
References 1. Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB (1994) An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoterluciferase construct. Anal Biochem 216(2):276–284 2. Abraham DJ, Eckes B, Rajkumar V, Krieg T (2007) New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma. Curr Rheumatol Rep 9(2):136–143 3. Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76(3):1274–1278 4. Blaauboer ME, Smit TH, Hanemaaijer R, Stoop R, Everts V (2011) Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts. Biochem Biophys Res Commun 404(1):23–27 5. Blumbach K, Zweers MC, Brunner G, Peters AS, Schmitz M, Schulz JN, Schild A, Denton CP, Sakai T, Fassler R, Krieg T, Eckes B (2010) Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts— role of TGFbeta1 levels and RhoA activity. J Cell Sci 123(Pt 22):3872–3883 6. Brown RA (2013) In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models? Exp Cell Res 319(16):2460–2469 7. Brown RA, Prajapati R, McGrouther DA, Yannas IV, Eastwood M (1998) Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J Cell Physiol 175(3):323–332 8. Butcher DT, Alliston T, Weaver VM (2009) A tense situation: forcing tumour progression. Nat Rev Cancer 9(2):108–122 9. Chaqour B, Goppelt-Struebe M (2006) Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins. FEBS J 273(16):3639–3649 10. Chiquet M (1999) Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 18(5):417–426
123
11. Fisher GJ, Varani J, Voorhees JJ (2008) Looking older: fibroblast collapse and therapeutic implications. Arch Dermatol 144(5):666–672 12. Gabrielli A, Avvedimento EV, Krieg T (2009) Scleroderma. N Engl J Med 360(19):1989–2003 13. Galie PA, Russell MW, Westfall MV, Stegemann JP (2012) Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-beta1. Exp Cell Res 318(1):75–84 14. Grinnell F, Petroll WM (2010) Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol 26:335–361 15. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12(9):2730–2741 16. Hinz B, Gabbiani G (2010) Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives. F1000 Biol Rep 2:78 17. Hoffman BD, Grashoff C, Schwartz MA (2011) Dynamic molecular processes mediate cellular mechanotransduction. Nature 475(7356):316–323 18. Holmes A, Abraham DJ, Sa S, Shiwen X, Black CM, Leask A (2001) CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem 276(14):10594–10601 19. Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Fujimoto M, Grotendorst GR, Takehara K (1996) Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. J Invest Dermatol 106(4):729–733 20. Kanazawa Y, Nomura J, Yoshimoto S, Suzuki T, Kita K, Suzuki N, Ichinose M (2009) Cyclical cell stretching of skin-derived fibroblasts downregulates connective tissue growth factor (CTGF) production. Connect Tissue Res 50(5):323–329 21. Kessler D, Dethlefsen S, Haase I, Plomann M, Hirche F, Krieg T, Eckes B (2001) Fibroblasts in mechanically stressed collagen lattices assume a ‘‘synthetic’’ phenotype. J Biol Chem 276(39):36575–36585 22. Leask A, Parapuram SK, Shi-Wen X, Abraham DJ (2009) Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio-marker of fibroproliferative disease? J Cell Commun Signal 3(2):89–94 23. Leung LY, Tian D, Brangwynne CP, Weitz DA, Tschumperlin DJ (2007) A new microrheometric approach reveals individual and cooperative roles for TGF-beta1 and IL-1beta in fibroblastmediated stiffening of collagen gels. Faseb J 21(9):2064–2073 24. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139(5):891–906 25. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D (1999) The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96(3):319–328 26. Munger JS, Sheppard D (2011) Cross talk among TGF-beta signaling pathways, integrins, and the extracellular matrix. Cold Spring Harb Perspect Biol 3(11):a005017 27. Pedersen JA, Swartz MA (2005) Mechanobiology in the third dimension. Ann Biomed Eng 33(11):1469–1490 28. Peters AS, Brunner G, Blumbach K, Abraham DJ, Krieg T, Eckes B (2012) Cyclic mechanical stress downregulates endothelin-1 and its responsive genes independently of TGFbeta1 in dermal fibroblasts. Exp Dermatol 21(10):765–770
Arch Dermatol Res 29. Reed RK, Rubin K (2010) Transcapillary exchange: role and importance of the interstitial fluid pressure and the extracellular matrix. Cardiovasc Res 87(2):211–217 30. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, Narins RG (1996) Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol 148(6):1915–1923 31. Saharinen J, Taipale J, Keski-Oja J (1996) Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. EMBO J 15(2):245– 253 32. Sarasa-Renedo A, Tunc-Civelek V, Chiquet M (2006) Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain. Exp Cell Res 312(8): 1361–1370 33. Schwartz MA (2010) Integrins and extracellular matrix in mechanotransduction. Cold Spring Harb Perspect Biol 2(12):a005066 34. Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T, Springer TA (2011) Latent TGF-beta structure and activation. Nature 474(7351):343–349 35. Singla V, Reiter JF (2006) The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313(5787): 629–633 36. Smith SM, Wastney ME, O’Brien KO, Morukov BV, Larina IM, Abrams SA, Davis-Street JE, Oganov V, Shackelford LC (2005) Bone markers, calcium metabolism, and calcium kinetics during extended-duration space flight on the mir space station. J Bone Miner Res 20(2):208–218
37. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3(5):349–363 38. Tomasek JJ, Hay ED (1984) Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels. J Cell Biol 99(2):536–549 39. Varga J, Abraham D (2007) Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 117(3):557–567 40. Wall ME, Banes AJ (2005) Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication. J Musculoskelet Neuronal Interact 5(1):70–84 41. Winter HH, Mours M (1997) Rheology of polymers near liquid– solid transitions. Adv Polym Sci 134:165–234 42. Wipff PJ, Rifkin DB, Meister JJ, Hinz B (2007) Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J Cell Biol 179(6):1311–1323 43. Wong M, Siegrist M, Goodwin K (2003) Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone 33(4):685– 693 44. Yamashiro T, Fukunaga T, Kobashi N, Kamioka H, Nakanishi T, Takigawa M, Takano-Yamamoto T (2001) Mechanical stimulation induces CTGF expression in rat osteocytes. J Dent Res 80(2):461–465
123
CONCISE COMMUNICATION
Cyclic mechanical strain induces TGFb1-signalling in dermal fibroblasts embedded in a 3D collagen lattice Andreas S. Peters • Georg Brunner Thomas Krieg • Beate Eckes
•
Received: 7 May 2014 / Revised: 30 September 2014 / Accepted: 7 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Many tissues are constantly exposed to mechanical stress, e.g. shear stress in vascular endothelium, compression forces in cartilage or tensile strain in the skin. Dermal fibroblasts can differentiate into contractile myofibroblasts in a process requiring the presence of TGFb1 in addition to mechanical load. We aimed at investigating the effect of cyclic mechanical strain on dermal fibroblasts grown in a three-dimensional environment. Therefore, murine dermal fibroblasts were cultured in collagen gels and subjected to cyclic tension at a frequency of 0.1 Hz (6 cycles/min) with a maximal increase in surface area of 10 % for 24 h. This treatment resulted in a significant increase in active TGFb1 levels, leaving the amount of total TGFb1 unaffected. TGFb1 activation led to pSMAD2-mediated transcriptional elevation of downstream mediators, such as CTGF, and an auto-induction of TGFb1, respectively. Keywords Mechanoregulation Tension Skin fibroblast CTGF
A. S. Peters (&) Department of Vascular and Endovascular Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany e-mail: [email protected] G. Brunner Department of Cancer Research, Fachklinik Hornheide, Mu¨nster, Germany T. Krieg B. Eckes Department of Dermatology, University of Cologne, Cologne, Germany
Introduction Cells and their surrounding tissues are constantly exposed to mechanical stress; many cellular functions rely on the existence of stress, some are even dependent on it, exemplified clearly by the action of gravity onto the musculoskeletal system since its absence causes bone and muscle atrophy in astronauts in space [36]. Other examples for the action of mechanical stress are compression forces in cartilage, shear stress in the vasculature, contraction forces in muscles and tendons as well as interstitial fluid pressure in tumour stroma [10, 29, 32]. They all influence cellular signalling and the properties of the respective tissues. Enhanced deposition and cross-linking of extracellular matrix (ECM) components surrounding a tumour is caused by ECM stiffness and is also involved in the development of malignancy and metastasis [24]. Enhanced stiffening also contributes to scarring and fibrosis and results in less pliable connective tissue. Alterations in stiffness are sensed by resident fibroblasts that display an activated phenotype and maintain the fibrotic response [12, 39]. With increasing age fibroblasts interact with a defective ECM leading to changes in skin homeostasis with altered production of ECM and proteases [11]. The conversion of external mechanical stimuli into intracellular biochemical signalling can be accomplished in various ways: the best characterised one is via integrins which cluster in focal adhesions and connect the ECM with intracellular actin microfilaments [10, 33]. Other transduction pathways include mechanosensitive ion-channels or cilia [35, 40]. Mechanically regulated genes have been investigated in various cell types and tissues, which are exposed to mechanical stress, like bone, endothelium and smooth muscle [17]. The extent of mechanical force in terms of
123
Arch Dermatol Res
tensile stress in healthy skin varies with anatomical location and can amount to several kPa. In fibrotic or desmoplastic tissue, forces can exceed 3 gPa [8]. Cultured fibroblasts can experience forces of \50 Pa if seeded into freely contracting collagen gels and up to [1 gPa if plated in monolayer cultures (ML) [14, 23]. Collagen I (COL I) is the most abundant connective tissue constituent in the skin. Hence, characterization of cells, cultured in a three-dimensional collagen matrix, is a well-established method to mimic the in vivo situation [3]. If attached to the perimeter of the well, the collagen lattice is prevented from contraction leading to a self-generated tensional force acting on the cells. Under these conditions human skin fibroblasts differentiate into a-smooth muscle actin (aSMA) positive myofibroblasts showing a bipolar morphology and are characterized by increased ECM synthesis and production of transforming growth factor b (TGFb) and connective tissue growth factor (CTGF), among other profibrotic factors [7, 21, 37, 38]. Hence, this culture model reflects conditions similar to those which are met in fibrotic and desmoplastic tissues [2, 16]. A key protein secreted by myofibroblasts and other mesenchymal cells in fibrosis is CTGF [22]. It has been demonstrated that the gene expression of CTGF is increased in fibrotic skin disorders [19]. Mechanical stress affects CTGF expression in a cell type- and stress-specific way: tension exerted on chondrocytes and bone causes an increase in CTGF, while fluid shear stress in endothelial cells leads to a reduction [9, 43, 44]. CTGF is most potently induced by TGFb1 and endothelin-1 (ET-1), although independent regulation has also been reported [18]. Production of active TGFb1 follows a complex pathway. First, the latent, inactive pro-form is produced consisting of the growth factor itself covalently attached to the latency associated peptide (LAP). Secretion of the proform is facilitated by binding of the latent LAP–TGFb1 complex to the latent TGFb-binding protein 1 (LTBP-1) which anchors the entire latent complex to the ECM via fibrillin and fibronectin [26, 31]. Activation of TGFb1 is then accomplished in the extracellular space either by proteolysis or via av integrins [25, 26, 34]. Our previous work showed that the application of cyclic mechanical stress to primary murine fibroblasts seeded as ML resulted in decreased CTGF and aSMA levels by a mechanism that is independent of TGFb1 but downstream of ET-1 [28]. This work provided a mechanistic explanation at the molecular level for the previously reported downregulation of CTGF and aSMA in human skin and lung fibroblasts [4, 20]. This study aimed at investigating the adaptive response of dermal fibroblasts to cyclic mechanical strain, focussing on production and activity of the profibrotic factor TGFb1
123
and corresponding downstream signalling. Fibroblasts were seeded in collagen lattices providing the cells with a 3D surrounding, similar to the in vivo situation—a culture model reflecting conditions which are met in fibrotic diseases.
Methods Culture of mouse dermal fibroblasts Primary murine dermal fibroblasts were isolated from the skin of newborn C57BL6 mice according to standard procedures and cultured in DMEM supplemented with 10 % foetal calf serum (FCS) as described [28]. Fibroblasts were used in passage 2–5. All animal protocols were approved by the local veterinary authorities (LANUV NRW 8.87-50.10.31.8.197). Application of cyclic strain All experiments were carried out using an FX-4000T Flexercell Tension Plus (Flexcell International Corporation, Hillsborough, NC, USA). Cells were seeded at a number of 0.5 9 106 in a total volume of 2 ml containing a final concentration of 0.3 mg/ml collagen I (Curacyte, Leipzig, Germany) as described [21]. The cell suspension was transferred to 6-well Tissue Train Plates (Flexcell International Corporation), each well having a flexible silicon membrane at the bottom and a circular foam ring attached to the perimeter of the well. The plates were incubated at 37 °C for 1 h to allow the collagen to polymerize. When using serum-free conditions, cells were washed thoroughly three times with PBS before mixing with the collagen solution, which was prepared free of FCS. Cyclic mechanical tension was applied in a sinusoidal pattern (0.1 Hz, 24 h) with a maximal equi-biaxial increase in surface area of 10 % (similar to [28, 32] ). Cells cultured with the identical collagen suspension in the same plates, but not exposed to cyclic mechanical strain, were used as controls. Isolation of RNA and RT-PCR Collagen gels were washed with PBS, transferred to a 2 ml Eppendorf tube containing 300 ll of RLT buffer (Qiagen, Hilden, Germany) and a 5-mm steel bead and homogenised using a Mixer Mill MM 300 (Retsch, Haan, Germany) at a frequency of 30 Hz for two minutes. Total RNA was isolated using the RNeasy Mini-Kit (Qiagen). Reverse transcription and semi-quantitative RT-PCR were performed as described [28] using the following primers: TGFb1 forward caa-cgc-cat-cta-tgagaa-aac-c, TGFb1 reverse aag-
Arch Dermatol Res
ccc-tgt-att-ccg-tct-cc (300 bp); CTGF forward cac-cta-aaatcg-cca-agcc, CTGF reverse gcc-atg-tctccg-tac-atc-ttc (271 bp); S26 forward aat-gtg-cag-ccc-att-cgc-tg, S26 reverse cttccg-tcc-tta-caa-aac-gg (324 bp). Intensity of bands corresponding to the analysed transcripts was quantified by densitometry using Image J (http://rsb.info. nih.gov/ij) and normalised to the intensity of S26 signals. Protein isolation and western blotting Collagen gels were washed with PBS and directly lysed by ultrasound in RIPA buffer [150 mM NaCl, 50 mM Tris– HCl, 1 % NP-40, 0.1 % SDS, 0.5 % Na-deoxycholate, 5 mM EDTA, 1 % Triton X-100 (Sigma-Aldrich, Steinheim, Germany), pH 8] containing protease- and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by SDS-PAGE, blotted and incubated with antibodies directed against pSMAD2 (Ser465 and Ser 467; Cell Signalling Technology, Frankfurt, Germany) in 5 % non-fat milk in TBS-T pH 7.6, followed by incubation with secondary HRP-conjugated antibodies (Dako, Glostrup, Denmark). Signals were detected using ECL (Thermo Scientific, Langenselbold, Germany). Membranes stained
Fig. 1 Cell morphology and gene regulation of murine dermal fibroblasts embedded in a 3D collagen lattice. Fibroblasts were seeded in collagen gels supplemented with 10 % FCS and exposed to cyclic mechanical tension (strained) or not (tethered) for 24 h. Cyclic stress created an increase in surface area of 10 % at a frequency of 0.1 Hz (96/min). Fibroblasts in both culture conditions showed actin stress fibres (F-actin, red; DAPI depicts nuclei, blue; magnification 9100).
with Ponceau S prior to antibody incubation were used as loading control. Determination of TGFb bioactivity TGFb1 levels were assessed in serum-free media supplemented with 0.1 % BSA (Low Endotoxin; PAA, Duesseldorf, Germany) that was conditioned for 24 h by fibroblasts. TGFb1 activity was determined using mink lung epithelial cells stably transfected with a luciferase reporter under the control of a truncated PAI-1 promoter (kind gift by Dr. Daniel B. Rifkin, New York University Medical Center) as described [1]. Immunohistochemistry Collagen gels were fixed in 4 % paraformaldehyde (PFA) in PBS for 15 min and processed as described [21] to visualise filamentous (F-) actin with Alexa Fluor 594 phalloidin (1:100 in 1 % BSA; Invitrogen, Eugene, OR, USA). Nuclei were counterstained with DAPI. Gels were transferred onto glass slides and mounted (Immu-Mount, Thermo Scientific). Photomicrographs were acquired using
a Fibroblast showed parallel alignment when strained or b were randomly oriented when left at rest. c Transcript levels for TGFb1 and CTGF were assessed by semi-quantitative RT-PCR. S26 RNA was used for normalisation. Bar graph shows quantitative analysis of signal intensity. Intensities of strained cultures (black) are displayed relative to tethered ones (grey) which were set at 1 (n = 3)
123
Arch Dermatol Res
an Olympus IX 71 microscope equipped with a DeltaVision system (Applied Precision, Issaquah, WA, USA). Viability assay Collagen gels were produced as described containing 0.5 9 106 cells and cultured for 24 h in DMEM supplemented with 10, 1 or 0 % FCS. Gels were washed thoroughly three times with PBS, stained with 0.4 % trypan blue solution (Sigma-Aldrich) and washed again three times with PBS. Living as well as dead cells were counted using a light microscope in four non-overlapping fields at 2009 magnification. Statistical analysis Statistical analysis was carried out using unpaired Student’s t test (in case of comparison of more than two groups prior analysis of variance was performed). Results are presented as mean ± standard deviation. A p value \0.05 was considered to be significant.
Results To assess the adaptive response of skin fibroblasts to cyclic mechanical tension, primary murine dermal fibroblasts
Fig. 2 TGFb1 bioavailability and -signalling in strained and tethered cultures. Fibroblasts were seeded in collagen gels and either exposed or not to cyclic stress for 24 h under serum-free conditions. TGFb1 bioactivity was determined in culture supernatants either directly or following heat activation reflecting the amount of active or total TGFb1, respectively. a Active TGFb1 is increased under strained (black, grey = tethered) conditions (**p = 0.00171) while total TGFb1 is unaltered. b The fraction of activated TGFb1 was even
123
were cultured in three-dimensional collagen lattices in complete medium and either exposed to equi-biaxial strain in a sinusoidal manner at 0.1 Hz (6/min) to attain an increase in surface area of 10 % (strained) or left at rest (tethered) for 24 h. Strained fibroblasts showed a parallel alignment (Fig. 1a) while tethered ones were randomly dispersed (Fig. 1b). In both conditions, fibroblasts assumed an extended bipolar shape and expressed pronounced stress fibres indicated by the staining (red) for F-actin (Fig. 1a, b). Application of cyclic mechanical tension induced a two or threefold increase in TGFb1 and CTGF transcript levels, respectively, as indicated by semi-quantitative RT-PCR (Fig. 1c). To determine whether the increased transcription levels resulted in elevated levels of secreted-bioactive TGFb1, serum-free collagen gels were subjected to cyclic mechanical strain for 24 h or left unstrained. The conditioned media were tested for the amount of total and active TGFb1 produced using the mink lung epithelial cell bioassay as described [1, 5, 28]. Supernatants of strained cultures showed a significant increase (**p = 0.00171) in active TGFb1 by 44 % compared to tethered controls while the amount of total activatable TGFb1 was unaffected (Fig. 2a). Consequently, the fraction of activated TGFb1 showed a further enhanced twofold increase with respect to strained fibroblasts (***p = 7.9331 9 10-5, Fig. 2b). To
further enhanced for strained (black, grey = tethered) cultures (***p = 7.9331 9 10-5). TGFb1 signalling was analysed in cell lysates by western blotting by detecting pSMAD2. Ponceau S staining of membranes after blotting indicates equal loading. c Elevated pSMAD2 signals were detected in strained compared to tethered cultures. Intensities of strained cultures (black) are displayed relative to tethered ones (grey) which were set at 1 (n = 3)
Arch Dermatol Res
many roundly rather than bipolar-shaped cells (Fig. 3b, c). However, in principle parallel orientation of fibroblasts in strained lattices was preserved (Fig. 3b).
Discussion
Fig. 3 Cell viability. a Fibroblasts were seeded in collagen gels with three different concentrations of FCS (10, 1 and 0 %) for 24 h. Collagen gels were incubated with 0.4 % trypan blue solution staining dead cells. Live as well as dead cells were counted using a light microscope in four non-overlapping fields at 9200 magnification. Results are presented as percentage live of total cells. Decreased FCS levels correlated with significant reduction of living fibroblasts (10 vs. 1 %: ***p 4.1943 9 10-6, 10 vs. 0 %: ***p 4.1912 9 10-5, 1 vs. 0 %: *p 0.0168; prior to analysis by t test, overall difference of means between the three groups was shown to be statistically significant by analysis of variance: ***p = 4.2109 9 10-7). b, c Fibroblasts were seeded in collagen gels and either exposed or not to cyclic stress for 24 h under serum-free conditions. Among reduced cell viability many cells appear rather roundly than bipolar shaped. However, the principle of parallel alignment of fibroblasts in strained cultures is preserved (F-actin, red; DAPI depicts nuclei, blue; magnification 9100)
assess whether the difference in bioactive TGFb1 is reflected by downstream signalling of TGFb1, pSMAD2 levels were determined in strained and resting cultures at the protein level by western immunoblotting. Levels of pSMAD2 were fourfold elevated in strained vs. tethered cultures (Fig. 2c), clearly confirming differential regulation of TGFb1 bioactivity by cyclic mechanical tension. To assess the effects of serum-free conditions in the 3Dculture system on primary murine skin fibroblasts, a viability assay was carried out using trypan blue solution to identify dead cells at three different concentrations of FCS (10, 1 and 0 %). Comparison of the three FCS concentrations revealed a highly statistically significant reduction in the amount of live cells with decreasing FCS concentration (Fig. 3a). Staining flexed as well as tethered cultures for F-actin (red) confirmed reduced cell viability, also showing
In this study we show that cyclic (equi-biaxial) mechanical tension induced TGFb1 signalling as well as increased expression of CTGF in primary murine skin fibroblasts. This result appears to contradict our previous report showing that myofibroblast differentiation was attenuated upon the action of tensile strain in ML [28]. However, ML cultures are devoid of bioactive TGFb1, whereas in 3D collagen lattices—both in strained as well as tethered— active TGFb1 was detected at 100–200 pg/ml, clearly exceeding the threshold at which fibroblasts are responsive. TGFb bioactivity was confirmed by enhanced phosphorylation of SMAD2. Under strained conditions amounts of total TGFb1 protein were unaltered compared to tethered controls. We were unable to assess whether TGFb1 activation is independent of protease activity; however, based upon prior studies demonstrating a stretch-induced conformation of LAP, that is independent of protease activity, we propose, that stretching of the collagen lattice may result in a protease-independent, stretch-induced activation of TGFb1 [42]. It has been shown before in rat mesangial cells cultured in monolayer and subjected to cyclic mechanical tension that TGFb1 was increased at both transcriptional as well as protein level (active and latent); interestingly in that study the fraction of active TGFb1 in stressed cultures remained unchanged compared to static controls [30]. Of note, regulation of latent TGFb activation appears to be cell- and stress-type specific, since uni-axially applied cyclic tension was reported to cause decreased SMAD-signalling in rat cardiac fibroblasts grown in a 3Dcollagen matrix [13]. Conducting experiments under serum-free conditions with the aim to exclude the interference of serum-derived growth factors and to reveal the bare effect of cyclic mechanical tension was difficult because of severely reduced cell viability. Although the culture of skin fibroblasts in collagen gels is closer to the in vivo situation by providing a 3D environment, which can be modulated by the application of force, the system falls short of several important features: Although collagen I is the most abundant ECM structural protein in skin, the 3D collagen lattice remains a single-component matrix offering adhesive properties and structural integrity but lacking other important components, e.g. elastin [27]. Also, in comparison to human dermis, the protein density is lower by a factor of approximately 103, while the water content with about 99 % is immensely high [6, 27]. In the 3D lattice,
123
Arch Dermatol Res
collagen monomers are laterally cross-linked to form fibres, which are merely entangled instead of covalently bound [41]. This can lead to so called non-affine deformation: the strain is not evenly distributed throughout the lattice due to fibre slippage, resulting in uniform dissipation of stress [27]. By contrast, ML does not deform in a nonaffine way, but rather deforms as a continuum [15]. To conclude, we show here for the first time that dermal fibroblasts grown in a collagen gel, providing them with a 3D surrounding mimicking the in vivo situation, and subjected to cyclic mechanical tension show elevated levels of bioactive TGFb1 and corresponding downstream signalling. By contrast, ML cultures provide an excellent tool to study effects based solely on biomechanical tension acting on fibroblasts without confounding influences of biologically active TGFb. Acknowledgments We thank Gabriele Scherr for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft through SFB 829 (to BE and TK) and KR 558/14.
References 1. Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB (1994) An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoterluciferase construct. Anal Biochem 216(2):276–284 2. Abraham DJ, Eckes B, Rajkumar V, Krieg T (2007) New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma. Curr Rheumatol Rep 9(2):136–143 3. Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76(3):1274–1278 4. Blaauboer ME, Smit TH, Hanemaaijer R, Stoop R, Everts V (2011) Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts. Biochem Biophys Res Commun 404(1):23–27 5. Blumbach K, Zweers MC, Brunner G, Peters AS, Schmitz M, Schulz JN, Schild A, Denton CP, Sakai T, Fassler R, Krieg T, Eckes B (2010) Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts— role of TGFbeta1 levels and RhoA activity. J Cell Sci 123(Pt 22):3872–3883 6. Brown RA (2013) In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models? Exp Cell Res 319(16):2460–2469 7. Brown RA, Prajapati R, McGrouther DA, Yannas IV, Eastwood M (1998) Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J Cell Physiol 175(3):323–332 8. Butcher DT, Alliston T, Weaver VM (2009) A tense situation: forcing tumour progression. Nat Rev Cancer 9(2):108–122 9. Chaqour B, Goppelt-Struebe M (2006) Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins. FEBS J 273(16):3639–3649 10. Chiquet M (1999) Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 18(5):417–426
123
11. Fisher GJ, Varani J, Voorhees JJ (2008) Looking older: fibroblast collapse and therapeutic implications. Arch Dermatol 144(5):666–672 12. Gabrielli A, Avvedimento EV, Krieg T (2009) Scleroderma. N Engl J Med 360(19):1989–2003 13. Galie PA, Russell MW, Westfall MV, Stegemann JP (2012) Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-beta1. Exp Cell Res 318(1):75–84 14. Grinnell F, Petroll WM (2010) Cell motility and mechanics in three-dimensional collagen matrices. Annu Rev Cell Dev Biol 26:335–361 15. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12(9):2730–2741 16. Hinz B, Gabbiani G (2010) Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives. F1000 Biol Rep 2:78 17. Hoffman BD, Grashoff C, Schwartz MA (2011) Dynamic molecular processes mediate cellular mechanotransduction. Nature 475(7356):316–323 18. Holmes A, Abraham DJ, Sa S, Shiwen X, Black CM, Leask A (2001) CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem 276(14):10594–10601 19. Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Fujimoto M, Grotendorst GR, Takehara K (1996) Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. J Invest Dermatol 106(4):729–733 20. Kanazawa Y, Nomura J, Yoshimoto S, Suzuki T, Kita K, Suzuki N, Ichinose M (2009) Cyclical cell stretching of skin-derived fibroblasts downregulates connective tissue growth factor (CTGF) production. Connect Tissue Res 50(5):323–329 21. Kessler D, Dethlefsen S, Haase I, Plomann M, Hirche F, Krieg T, Eckes B (2001) Fibroblasts in mechanically stressed collagen lattices assume a ‘‘synthetic’’ phenotype. J Biol Chem 276(39):36575–36585 22. Leask A, Parapuram SK, Shi-Wen X, Abraham DJ (2009) Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio-marker of fibroproliferative disease? J Cell Commun Signal 3(2):89–94 23. Leung LY, Tian D, Brangwynne CP, Weitz DA, Tschumperlin DJ (2007) A new microrheometric approach reveals individual and cooperative roles for TGF-beta1 and IL-1beta in fibroblastmediated stiffening of collagen gels. Faseb J 21(9):2064–2073 24. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139(5):891–906 25. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D (1999) The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96(3):319–328 26. Munger JS, Sheppard D (2011) Cross talk among TGF-beta signaling pathways, integrins, and the extracellular matrix. Cold Spring Harb Perspect Biol 3(11):a005017 27. Pedersen JA, Swartz MA (2005) Mechanobiology in the third dimension. Ann Biomed Eng 33(11):1469–1490 28. Peters AS, Brunner G, Blumbach K, Abraham DJ, Krieg T, Eckes B (2012) Cyclic mechanical stress downregulates endothelin-1 and its responsive genes independently of TGFbeta1 in dermal fibroblasts. Exp Dermatol 21(10):765–770
Arch Dermatol Res 29. Reed RK, Rubin K (2010) Transcapillary exchange: role and importance of the interstitial fluid pressure and the extracellular matrix. Cardiovasc Res 87(2):211–217 30. Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, Narins RG (1996) Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells. Am J Pathol 148(6):1915–1923 31. Saharinen J, Taipale J, Keski-Oja J (1996) Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. EMBO J 15(2):245– 253 32. Sarasa-Renedo A, Tunc-Civelek V, Chiquet M (2006) Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain. Exp Cell Res 312(8): 1361–1370 33. Schwartz MA (2010) Integrins and extracellular matrix in mechanotransduction. Cold Spring Harb Perspect Biol 2(12):a005066 34. Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T, Springer TA (2011) Latent TGF-beta structure and activation. Nature 474(7351):343–349 35. Singla V, Reiter JF (2006) The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313(5787): 629–633 36. Smith SM, Wastney ME, O’Brien KO, Morukov BV, Larina IM, Abrams SA, Davis-Street JE, Oganov V, Shackelford LC (2005) Bone markers, calcium metabolism, and calcium kinetics during extended-duration space flight on the mir space station. J Bone Miner Res 20(2):208–218
37. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3(5):349–363 38. Tomasek JJ, Hay ED (1984) Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels. J Cell Biol 99(2):536–549 39. Varga J, Abraham D (2007) Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 117(3):557–567 40. Wall ME, Banes AJ (2005) Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication. J Musculoskelet Neuronal Interact 5(1):70–84 41. Winter HH, Mours M (1997) Rheology of polymers near liquid– solid transitions. Adv Polym Sci 134:165–234 42. Wipff PJ, Rifkin DB, Meister JJ, Hinz B (2007) Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J Cell Biol 179(6):1311–1323 43. Wong M, Siegrist M, Goodwin K (2003) Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone 33(4):685– 693 44. Yamashiro T, Fukunaga T, Kobashi N, Kamioka H, Nakanishi T, Takigawa M, Takano-Yamamoto T (2001) Mechanical stimulation induces CTGF expression in rat osteocytes. J Dent Res 80(2):461–465
123
