Small Mechanical Strains Selectively Suppress Matrix Metalloproteinase-1 Expression by Human Vascular Smooth Muscle Cells*

Mechanical forces and biochemical stimuli may interact to regulate cellular responses. In this study, we tested the hypothesis that very small mechanical strains interact with growth factors in the regulation of matrix metalloproteinase (MMP)-1. Human vascular smooth muscle cells (VSMCs) were cultured on a precoated silicone membrane in a device that imposes a highly uniform biaxial strain. VSMCs cultured on fibronectin were treated with cyclic 1-Hz strains of 0, 1, or 4%, and MMPs were assayed by Western analysis or gelatin zymography. Small strains did not induce MMP-1 in VSMCs, but strain was a potent inhibitor of platelet-derived growth factor (PDGF)- or tumor necrosis factor-α-induced synthesis of MMP-1. In contrast, MMP-2 and TIMP-2 levels were not changed by PDGF and/or mechanical strain. VSMCs strained on the 120-kDa chymotryptic fragment of fibronectin or RGD peptides suppressed PDGF-induced expression of MMP-1, indicating that this effect is not mediated by the heparin-binding domain or connecting segment-1 of fibronectin. Northern analysis of ets-1, a transcriptional activator of MMP-1 expression, showed that strain down-regulated ets-1expression, whereas c-fos expression was augmented. Thus, small deformations can selectively suppress MMP-1 synthesis by VSMCs, demonstrating the exquisite sensitivity of the cell to mechanical stimuli.

In the vessel wall as well as in other tissues, adaptation occurs through constant cellular migration, proliferation, and death and extracellular matrix synthesis and degradation. In stable normal tissues, the rates of these processes are so slow that they may appear almost dormant, whereas in injured or repairing tissues, the processes may all be greatly accelerated. In any wound repair, it is critical that the repaired tissue be sufficiently strong to withstand mechanical forces the tissue may experience. However, while the importance of biochemical mediators such as cytokines and growth factors in tissue repair and homeostasis is clear (1)(2)(3), how mechanical forces may regulate tissue structure and repair is less understood. The regulation of the wound repair response by mechanical forces is particularly relevant to the cardiovascular system, which must withstand large dynamic fluctuations in strain.
Several studies have shown that mechanical forces exert important regulatory effects on vascular smooth muscle cells (VSMCs), 1 including increased collagen synthesis and cell growth by mechanical strain (4 -6). Wilson et al. (7,8) have demonstrated that mechanical strain stimulates a mitogenic response in rat VSMCs through induction of platelet-derived growth factor (PDGF), and this induction is regulated by specific extracellular matrix interactions. Thus, VSMCs may sense mechanical stimuli (mechanotransduction) and change arterial structure.
One way VSMCs can change arterial structure is through the matrix metalloproteinases (MMPs). The MMPs are members of a family of enzymes that digest specific components of the extracellular matrix and may play a critical role in tissue repair and remodeling. The enzymatic activity of MMPs is regulated at several levels, including transcription; for example, cytokines such as IL-1, TNF-␣, and PDGF induce secretion of MMP proenzymes (9,10). These latent proenzymes can then be activated in the extracellular space (11,12). Finally, the active MMPs may be inhibited by TIMPs (tissue-type inhibitors of matrix metalloproteinase), specific endogenous inhibitors of the MMPs. Several MMP promoters contain the 12-O-tetradecanoylphorbol-13-acetate response element, which binds AP-1, transcription factor dimeric combinations of c-Fos and c-Jun, as well as polyoma enhancer activator sites (PEA3), which bind the Ets family of transcription factors (13,14).
Several recent lines of evidence suggest that mechanotransduction through the extracellular matrix may regulate secretion of MMPs. First, James et al. (15) reported that direct mechanical wounding of a cellular monolayer of VSMCs induces MMP expression. Second, perturbing the cytoskeleton of rabbit synovial fibroblasts induces MMP expression (16). Third, Huhtala et al. (17) have demonstrated that domains of fibronectin (FN) may interact with different integrin subunits to either induce or suppress collagenase (MMP-1) expression in rabbit synovial fibroblast cells. Finally, we and others (18,19) have observed that, in vivo, MMP-1 is overexpressed at sites of mechanical overload in the diseased artery.
Using a mechanical deformation device that applies a highly uniform biaxial strain field over the culture substrate, we explored the hypothesis that mechanical deformations regulate MMP-1 secretion by VSMCs. Surprisingly, we found that very small strains do not induce MMP-1, but abolish induction of MMP-1 by PDGF or TNF-␣, demonstrating that mechanical stimuli may potently interact with biochemical signals in regulating extracellular matrix metabolism. and Ham's F-12 were obtained from BioWhittaker, Inc. Dulbecco's phosphate-buffered saline solution, Hanks' salt  solution, fibronectin, 120-kDa chymotryptic fibronectin fragment,  GRGDSP peptide, phorbol 12-myristate 13-acetate, and other materials  required for tissue culture were purchased from Life Technologies, Inc. Ovalbumin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Tris, glycine, sodium chloride, and sodium dodecyl sulfate were obtained from Sigma. PDGF-BB, IL-1␣, and TNF-␣ were from Collaborative Biomedical Products (Bedford, MA), and collagen was from Cell Biomaterials (Palo Alto, CA). Prestained low molecular mass markers and acrylamide gel buffer were purchased from Bio-Rad. [␣-32 P]dCTP (3000 Ci/mmol) was purchased from NEN Life Science Products.

Materials-DMEM
Culture of VSMCs-Human VSMCs were derived from explants of discarded portions of saphenous vein after coronary bypass surgery from Brigham and Women's Hospital. VSMCs were maintained in DMEM, 10% fetal calf serum, and 1% penicillin/streptomycin sulfate. These conditions are selective for growth of smooth muscle cells over endothelial cells (20). VSMCs were maintained at 37°C in 5% CO 2 up to passages 6 -7 for experiments. Approximately 50% of cells using these techniques stained positively for ␣-actin.
Mechanical Strain Device and Preparation of Cells-Mechanical deformation was applied to a thin and transparent membrane on which cells were cultured, an approach that produces controlled cellular strain as well as visualization of cells. This device provides a nearly homogeneous biaxial strain profile, i.e. strains that are equal at all locations on the membrane and in all directions (21). An advantage of this device over some commonly used systems is that it eliminates locations on the substrate that have very high strains (20 -30%) in one direction. Each culture dish consists of a plastic (Kynar) cylinder and a circular silicone elastometric membrane, which is the culture surface. The membrane undergoes cyclic tensile deformation as the platen assembly moves sinusoidally with a frequency and amplitude derived by the motor speed and the cam size, respectively. We have previously measured membrane strains with a high resolution video device (22); the cams used for this study gave strains of 1.0 Ϯ 0.1% and 4.2 Ϯ 0.1% (n ϭ 18 different locations for each).
The cell culture silicone membrane itself supports negligible adhesion of VSMCs. In these experiments, three different methods of supporting adhesion were used: precoating with intact FN, 120FN (the 120-kDa chymotryptic peptide of FN that contains the RGD sequence), and RGD peptide coupled to ovalbumin or collagen. When VSMCs were plated on membranes precoated with equivalent concentrations (2 g/ ml) of FN, 120FN, and ovalbumin-coupled RGD peptide, there was no morphological difference in VSMCs.
For the preparation of VSMCs to be subjected to mechanical strain, autoclaved membrane dishes were coated with 2 g/ml FN in 13 ml of Hanks' solution for 6 -12 h at 4°C and then washed twice with 10 ml of phosphate-buffered saline. VSMCs were plated on the coated membrane dish at a density of 400,000 cells/dish in 13 ml of DMEM containing 10% FCS and incubated 16 -24 h. For culturing VSMCs on the collagen, 50 g/ml collagen was used. VSMCs were then washed four times with 10 ml of Hanks' solution to remove residual serum and incubated with 10 ml of serum-free "IT" medium (equal volumes of DMEM and Ham's F-12 supplemented with 1 mol/liter insulin and 5 g/ml transferrin) for 48 h. Before mechanical strain or cytokine stimulation, 10 ml of fresh IT medium was exchanged. Mechanical strain was then applied at a specified constant frequency and amplitude, and control dishes received no mechanical strain.
RGD Peptide Coupling-The coupling of RGD (Arg-Gly-Asp) peptides with ovalbumin was performed as described by Haugen et al. (23). Equal amounts (by weight) of GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptides and ovalbumin were solubilized and mixed with a 10-fold mass of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolved in deionized water. After mixing overnight in the dark at 4°C, the coupled samples were extensively dialyzed against phosphatebuffered saline to remove excess carbodiimide and uncoupled peptides (10,000 exclusion membrane, Spectrum Medical Industries, Inc., Houston, TX).
Western Analysis-Conditioned media were concentrated by Centricon-10 miniconcentrators (Amicon, Inc.). Samples were loaded on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane in 25 mM Tris base (pH 8.5), 0.2 M glycine, and 20% methanol. The nitrocellulose membrane was blocked with 5% nonfat dried milk in TBS washing buffer (20 mM Tris base (pH 7.6), 137 mM NaCl, and 0.1% Tween 20) for 2 h. For the detection of collagenase I (MMP-1), the membrane was incubated with 1:2000 diluted rabbit anti-human colla-genase polyclonal antibody (generous gift of Merck Research Laboratories) for 1 h at 37°C and washed with TBS washing buffer for 30 min. The secondary antibody, goat anti-rabbit IgG coupled to peroxidase, was diluted 1:4000 and incubated with the membrane for 30 min. After washing with TBS washing buffer for 30 min, the membrane was developed by the enhanced chemiluminescence method (ECL, Amersham Life Science, Inc.). For the detection of TIMP-1 and TIMP-2, 1:4000 diluted rabbit anti-human TIMP-1 polyclonal antibody and 1:1000 diluted rabbit anti-human TIMP-2 polyclonal antibody (gift of Amgen) were used, respectively. Quantitation of Western analyses was performed by scanning densitometry using the Optimas 5.2 software package (Optimas Corp., Bothell, Washington). Quantitative data are presented as the mean Ϯ S.D. from three independent experiments.
Northern Analysis-Total RNA was isolated by the guanidium thiocyanate and phenol chloroform method (24). The full-length 1. The membrane was washed with 2ϫ SSC and 0.05% SDS solution for 30 -40 min (three times at room temperature) and with 0.1ϫ SSC and 0.1% SDS solution with continuous shaking at 50°C for 40 min. The membrane was exposed to x-ray film at Ϫ80°C.

RESULTS
Strain Regulates MMP-1 Secretion by VSMCs-We first evaluated the effect of mechanical strain on MMP-1 synthesis by VSMCs plated on intact FN. When cells were subjected to 0, 1, and 4% cyclic mechanical strains at 1 Hz in multiple experiments, mechanical strain did not induce MMP-1 expression, whereas PDGF-BB induced MMP-1 (9.2 Ϯ 1.1-fold, n ϭ 3). Surprisingly, 1% cyclic mechanical strain suppressed 76 Ϯ 24% (n ϭ 3) of PDGF-induced MMP-1 expression, and 4% strain suppressed 89 Ϯ 15% of MMP-1 expression (Fig. 1A). In these studies, no morphological changes in cells were detected following strains of 1 or 4%, and we have previously demonstrated that cellular injury and fibroblast growth factor-2 release occur only at strains above 10% in VSMCs (22). Thus, these findings suggested that specific regulation and not merely cellular injury caused suppression of MMP-1 synthesis by mechanical deformations under these conditions.
Effect of Strain on MMP-1 Regulation by VSMCs on 120FN and RGD Peptides-Huhtala et al. (17) have demonstrated that the 120-kDa chymotryptic fragment of fibronectin (120FN), which contains the RGD sequence but does not contain the connecting segment-1 of fibronectin, can induce MMP-1 secretion by rabbit synovial fibroblasts. Thus, our finding that small mechanical strains on intact FN can suppress cytokine-induced MMP-1 secretion raised the hypothesis that mechanical strains suppress MMP-1 secretion through non-RGD domains of FN. To explore this hypothesis, we plated VSMCs on 2 g/ml 120FN, a concentration sufficient to support equivalent amounts of adhesion of cells as intact FN coating of a membrane. In contrast to previous reports on rabbit synovial fibroblasts, plating VSMCs on 120FN did not induce MMP-1, nor did mechanical strain induce MMP-1 expression by VSMCs on 120FN. PDGF induced MMP-1 in VSMCs plated on 120FN (5.2 Ϯ 1.9-fold, n ϭ 3), and 1 and 4% cyclic mechanical strains suppressed PDGF-induced MMP-1 by 37 Ϯ 15% and 50 Ϯ 23%, respectively (Fig. 1B). This experiment indicated that strain did not inhibit MMP-1 synthesis through adhesion to the connecting segment-1 region of FN.
In addition, we studied VSMCs plated on ovalbumin-coupled RGD peptide (2 g/ml). MMP-1 expression by VSMCs on RGD peptides was similar to that by VSMCs on intact FN or 120FN (7.0 Ϯ 3.3-fold, n ϭ 3). Similar to the effects of FN and 120FN, PDGF induced MMP-1 expression on RGD peptides, but 1 and 4% mechanical strains also suppressed 52 Ϯ 28% and 72 Ϯ 28% of PDGF-induced MMP-1 synthesis, respectively (Fig. 1C). These data further support the finding that suppression of MMP-1 by small mechanical strains does not require the connecting segment-1 domain of FN and that mechanical strain through the RGD domain is sufficient for this effect.
Effect of Strain on MMP-2 and MMP-9 -Although many MMPs have similar promoter elements, expression of the gelatinases, particularly MMP-2, may be independent of MMP-1 expression (25). Therefore, we evaluated the expression of MMP-2 (72-kDa gelatinase) and MMP-9 (92-kDa gelatinase) by VSMCs in response to mechanical stimuli and PDGF treatment. Neither mechanical strain nor PDGF treatment changed constitutive MMP-2 expression (Fig. 2A). VSMCs plated on 120FN or ovalbumin-coupled RGD peptide also expressed similar levels of MMP-2 regardless of PDGF treatment or mechan-ical strain (Fig. 2, B and C). We also found MMP-9 activity in VSMCs plated on FN, but the activity was very weak compared with MMP-2 activity. Neither PDGF treatment nor mechanical strain increased MMP-9 expression ( Fig. 2A). On 120FN or ovalbumin-coupled RGD peptides, MMP-9 induction was not observed compared with intact FN, and neither PDGF treatment nor mechanical strain increased MMP-9 expression (Fig.  2, B and C). These results eliminated the possibility that all MMPs were down-regulated by mechanical strain.
In contrast to the effects of mechanical strain on MMP-1 and TIMP-1 expression, the expression of TIMP-2, the specific inhibitor of MMP-2, by VSMCs cultured on FN was not changed by PDGF treatment or mechanical strain (Fig. 4A). When VSMCs were cultured on 120FN or ovalbumin-coupled RGD

FIG. 1. Effect of strain on MMP-1 synthesis by VSMCs on intact FN (A), 120FN (B), or RGD peptides (C).
VSMCs were plated on 2 g/ml FN, 120FN, or RGD peptides coupled to ovalbumin in DMEM containing 10% FCS for 24 h. After serum deprivation for 48 h, cells were subjected to 0, 1, and 4% cyclic mechanical strains (1 Hz) for 24 h with (ϩ) or without (Ϫ) PDGF-BB (5 ng/ml), and media were analyzed by Western analysis with anti-human MMP-1 polyclonal antibody. When cells were plated on FN or 120FN, even 1% strains suppressed MMP-1 synthesis in the presence of PDGF. The upper bands seen in B correspond to the zymogen forms of MMP-1.
Strain and MMP-1 Regulation by Human VSMCs Cultured on Collagen-Extracellular matrix components may influence both MMP-1 expression and mechano-responsiveness (8,27). VSMCs were plated on 50 g/ml collagen, a concentration optimal for VSMC adherence to the silicone membrane. Similar to the response of VSMC plated on fibronectin, strain did not induce MMP-1 expression, but suppressed MMP-1 synthesis by PDGF induction even at 1% amplitude (Fig. 6).
Northern Analysis-To explore potential mechanisms of regulation of MMP-1 and TIMP synthesis by mechanical strain, VSMCs cultured on fibronectin were subjected to 4% strain in the presence of PDGF after 2 days of serum deprivation. Northern analysis for MMP-1 showed that 4% strain suppressed steady-state MMP-1 mRNA levels, whereas TIMP-1 mRNA levels were not appreciably changed (Fig. 7).
Mechanical strains can induce c-Fos, a component of the AP-1 complex that can promote transcription of MMP-1. Phosphorylation of c-Jun by JNK (c-Jun NH 2 -terminal kinase) is a signaling mechanism stimulated by physical stress (28) such as UV irradiation, heat shock, and several cytokines (29 -31); increased JNK activity is another mechanism by which mechanical strain may stimulate AP-1 activity (32). To explore the potential mechanism of MMP-1 suppression by strain, cultured VSMCs on FN were subjected to 4% strain. Induction of c-fos was observed after 30 min of strain (Fig. 8). In addition, JNK activity was not significantly affected (data not shown). Members of the Ets family of transcriptional activators also regulate MMP-1 expression by interacting with the AP-1 complex (33)(34)(35). Northern analysis of ets-1 demonstrated that strain suppressed ets-1 expression by PDGF (Fig. 8). Therefore, the suppression of MMP-1 by small strains may be related to suppression of ets-1, but not suppression of c-fos. DISCUSSION Previous studies have suggested that mechanical injury to cells and cytoskeletal deformation stimulate MMP synthesis and secretion. In addition, activation of components of the AP-1 complex, a potent positive regulator of MMP-1 transcription, can occur with mechanical deformation of cells. Thus, we initially anticipated that small mechanical deformations of VSMCs would induce MMP-1 synthesis. In addition, we hypothesized that this induction would be independent of, or synergistic with, biochemical stimulation of MMP-1 synthesis, such as by PDGF. Surprisingly, we found that small mechanical deformations do not induce MMP-1 synthesis, but potently suppress MMP-1 synthesis by PDGF. These findings indicate that small mechanical deformations may be a powerful influence on cellular control of extracellular matrix degradation.

FIG. 5. Effect of strain on IL-1␣-and TNF-␣-induced MMP-1 synthesis by VSMCs.
VSMCs were plated on 2 g/ml FN in DMEM containing 10% FCS for 24 h. After serum deprivation for 48 h, cells were subjected to 0 and 4% cyclic mechanical strains (1 Hz) for 24 h with (ϩ) or without (Ϫ) 10 ng/ml IL-1␣ or TNF-␣. Media were analyzed by Western analysis with anti-human MMP-1 polyclonal antibody. PDGF-BB (5 ng/ml) was used as positive control for MMP-1 induction. ment 120FN containing the RGD domain induces MMP-1 synthesis by rabbit synovial fibroblasts. In addition, the ability of non-RGD domains of fibronectin to suppress MMP-1 synthesis appears to be mediated by interactions with ␣ 4 ␤ 1 -integrins rather than the RGD-binding classical fibronectin receptor, ␣ 5 ␤ 1 . In contrast, in these experiments, when human VSMCs were plated on fibronectin, 120FN, or RGD peptides, MMP-1 was not induced. The difference between these results may be due to differences between rabbit synovial fibroblasts and hu-man vascular smooth muscle cells. For example, human VSMCs have very little ␣ 4 ␤ 1 by immunoprecipitation (37), so perhaps non-RGD domains of fibronectin cannot regulate MMP-1 synthesis by VSMCs. The similarity of the strain effect on MMP-1 regulation by VSMCs on collagen implies that strain itself might be a powerful regulator of MMP-1 synthesis regardless of the type of extracellular matrix component. The matrix, integrins, plasma membrane, cytoskeleton, and nucleus are interconnected, and mechanical deformation of any cellular component may lead to a change in shape of another cellular component (38 -41). Therefore, it is possible that integrins provide anchors to link a cytoskeleton or nuclear mechanotransduction mechanism to the extracellular space. Studies by Wilson et al. (8) suggest that ␣ v -integrins may regulate mechanotransduction, even when cells are adherent to matrices other than fibronectin or vitronectin. Further studies will be necessary to determine if mechanical strain through specific ␣or ␤-integrin subunits can regulate MMP synthesis.
One of the advantages of the mechanical strain device used in this experiment is that strains are precise and highly uniform. In many other types of cell-stretching devices, regions of the cell culture substrate have very high strains compared with the mean strain. For example, in one commonly used device, the strain can range from 0 to 33% depending on the location and orientation of the cell (21). We have previously demonstrated that cellular injury and fibroblast growth factor-2 release occur only when VSMCs are exposed to membrane strains higher than 10% (22). Imposing small strains with this cellular deformation device prevents confusing paracrine effects (such as fibroblast growth factor-2 release) from non-uniform strain profiles. In addition, the precise strains imposed in these experiments eliminate the possibility that suppression of MMP-1 by strains as small as 1% was due to cellular injury. Furthermore, the experiments demonstrating that MMP-2 and TIMP-2 syntheses are not suppressed by small strains indicate that the suppression of MMP-1 is selective.
These experiments explored some potential mechanisms of the suppression of MMP-1 synthesis by strain. Strain augmented c-fos expression in the presence of PDGF, but suppressed ets-1 expression. However, there are at least 30 members of the Ets-1 family, and mechanisms other than ets-1 down-regulation could play a major role. Westermarck et al. (42) reported that PU.1, a member of the Ets family, can suppress MMP-1 synthesis. In our studies, however, we have not found PU.1 expression by VSMCs. Our studies suggest that direct effects of the PDGF receptor are not primarily responsible for the effects of strain, as strain could also suppress MMP-1 induction by TNF-␣. We have evaluated cytoskeletal changes under these conditions using rhodamine-phalloidin staining, which demonstrated no clear effect of strains on the actin cytoskeleton (data not shown); however, we cannot exclude important cytoskeletal changes as a mechanism for mechanotransduction under these conditions. Previous reports have described mechanically mediated synthesis and release of PDGF from rat VSMCs subjected to mechanical deformation (7). In pilot studies, we have not observed induction of the PDGF-A or PDGF-B genes in human VSMCs exposed to the small mechanical deformations used in the present study. 2 It is possible that species-specific responses or differences in the mechanical strain devices could explain these differences. Furthermore, mechanically mediated release of PDGF would not explain why small strains can suppress the induction of MMP-1 by exogenous PDGF. FIG. 6. Effect of strain on MMP-1 synthesis by VSMCs cultured on collagen. VSMCs were cultured on 50 g/ml collagen in DMEM containing 10% FCS for 24 h. After serum deprivation for 48 h, cells were subjected to 0, 1, and 4% cyclic mechanical strains (1 Hz) for 24 h with (ϩ) or without (Ϫ) PDGF-BB (5 ng/ml), and media were analyzed by Western analysis with anti-human MMP-1 polyclonal antibody.

FIG. 7. Effect of strain on MMP-1 and TIMP-1 mRNA synthesis.
VSMCs were cultured on intact FN (2 g/ml) in DMEM containing 10% FCS for 24 h. After serum deprivation for 48 h, 0 and 4% cyclic mechanical strains were imposed for 0, 1, 3, 6, and 12 h with (ϩ) PDGF-BB (5 ng/ml) treatment. Total RNA was isolated at each time point, and MMP-1 and TIMP-1 were analyzed by Northern analysis. The ethidium bromide-stained 18 S ribosomal subunit is also shown.

FIG. 8. Effect of strain on the expression of ets-1 and c-fos.
VSMCs were cultured on intact FN (2 g/ml) in DMEM containing 10% FCS for 24 h. After serum deprivation for 48 h, 0 and 4% cyclic mechanical strains were imposed for 0, 0.5, 1, 6, 12, and 24 h with (ϩ) PDGF-BB (5 ng/ml) treatment. Total RNA was isolated at each time point, and ets-1 and c-fos were analyzed by Northern analysis. The ethidium bromide-stained 18 S ribosomal subunit is also shown.
In vivo, MMPs are frequently found in tissues undergoing repair or remodeling, such as high stress locations of the human atherosclerotic lesion (18,19,43). In these circumstances, cytokines and growth factors are generally abundant and regulate the repair process. However, cells in a repairing tissue must also produce a new tissue that can withstand mechanical forces imposed on that tissue, such as the constant tension of the skin or the pulsatile forces of blood pressure in the artery. Although strains of only 1% are imperceptible to the eye, this magnitude of cellular deformation was sufficient to suppress MMP-1 synthesis. This demonstrates the exquisite sensitivity of the cell to mechanical stimuli and the powerful influence of mechanical forces on responses to growth factors. Unraveling mechanotransduction mechanisms will likely reveal complex interactions with other systems of signal transduction.