©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanical Strain Induces pp60 Activation and Translocation to Cytoskeleton in Fetal Rat Lung Cells (*)

(Received for publication, October 16, 1995; and in revised form, December 15, 1995)

Mingyao Liu Yi Qin Jason Liu A. Keith Tanswell Martin Post (§)

From the Medical Research Council Group in Lung Development and the Neonatal Research Division, Hospital for Sick Children Research Institute, Department of Pediatrics, University of Toronto, Toronto M5G 1X8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that mechanical strain-induced fetal rat lung cell proliferation is transduced via the phospholipase C--protein kinase C pathway. In the present study, we found that protein-tyrosine kinase activity of fetal lung cells increased after a short period of strain, which was accompanied by tyrosine phosphorylation of proteins of 110-130 kDa. Several components of this complex were identified as pp60substrates. Strain increased pp60 activity in the cytoskeletal fraction, which coincided with a shift in subcellular distribution of pp60 from the Triton-soluble to the cytoskeletal fraction. Strain-induced pp60 translocation did not appear to be mediated via the focal adhesion kinase-paxillin pathway. In contrast, strain increased the association between pp60 and the actin filament-associated protein of 110 kDa. Preincubation of cells with herbimycin A, a tyrosine kinase inhibitor, abolished strain-induced phospholipase C-1 tyrosine phosphorylation and its coimmunoprecipitation with pp60. It also inhibited strain-induced DNA synthesis. These results suggest that activation of pp60 is an upstream event of the phospholipase C--protein kinase C pathway that may represent an important mechanism by which mechanical perturbations are converted to biological reactions in fetal lung cells.


INTRODUCTION

All tissues in the body are subjected to physical forces originating either from tension, created by cells themselves, or from the environment(1) . The role of mechanical force as an important regulator of structure and function of mammalian cells has recently been recognized for many tissues and cell types(2) . Although the biological responses of cells to physical forces vary, physical stimuli have to be sensed by cells and transmitted through intracellular signal transduction pathways. In response to mechanical stimulation, cells may generate a variety of second messengers and activate different phospholipases, protein kinases, and other signal transduction-related enzymes, depending upon the tissue and cell type, developmental stage, the manner and period of physical forces applied, and environmental conditions(2, 3) .

Respiration is a unique feature of the lung. Physical forces, derived from breathing, play an important role in regulating lung structure, function, and metabolism(4) . ``Breathing movements'' can be observed by sonography in the human fetus as early as 10 weeks of gestation. There are several lines of evidence from physiological studies and clinical observations to suggest that fetal breathing movements play an important role in lung growth(5, 6) . We have shown that an intermittent strain regimen that simulates fetal breathing movements enhanced DNA synthesis and cell division of fetal rat lung cells maintained in organotypic culture(7, 8, 9) . We have also observed a dramatic increase in the intracellular concentrations of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, after a short period of strain. These increases in inositol 1,4,5-trisphosphate and diacylglycerol were accompanied by an increased tyrosine phosphorylation of phospholipase C-1 (PLC-1) (^1)and protein kinase C (PKC) activity. Strain-induced PKC activation and DNA synthesis were blocked by PLC and PKC inhibitors(9) . These results suggest that mechanical strain-induced fetal lung cell proliferation is mediated through the PLC--PKC pathway. However, the mechanism by which strain-induced cytoskeleton deformation is converted to biochemical reactions remains unknown.

Herein, we report that mechanical strain of fetal rat lung cells induced a rapid activation and translocation of pp60 from the Triton-soluble to the cytoskeletal fraction. Strain-induced pp60 translocation appears to be mediated via the actin filament-associated protein of 110 kDa (AFAP-110). Preincubation of cells with a protein-tyrosine kinase (PTK) inhibitor, herbimycin A, blocked strain-induced PLC-1 tyrosine phosphorylation and its association with pp60 as well as strain-induced DNA synthesis. These results suggest that strain-induced PTK activation is an upstream event of the PLC--PKC pathway.


EXPERIMENTAL PROCEDURES

Materials

Pregnant Wistar rats (200-250 g) were obtained from Charles River (St. Constant, Quebec, Canada). Eagle's minimal essential medium and antibiotics were purchased from Life Technologies, Inc. Fetal bovine serum, PTK (Src family) assay kit, and polyclonal anti-phosphotyrosine, polyclonal and monoclonal anti-pp60, monoclonal anti-focal adhesion kinase (pp125), monoclonal anti-pp80/85 (cortactin), monoclonal anti-pp120, monoclonal anti-PLC-1, and polyclonal anti-GTPase-activating protein antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-AFAP-110 and monoclonal anti-pp130 antibodies were generous gifts of Dr. J. T. Parsons (University of Virginia). Monoclonal anti-paxillin antibody was from Transduction Laboratories (Lexington, KY). PTK substrate peptide (RR-SRC) was from Life Technologies, Inc. (Burlington, Ontario, Canada). Gelfoam sponges were from Upjohn (Toronto, Ontario). Protein assays were performed using a kit from Bio-Rad. All other chemicals were from Sigma.

Strain of Fetal Lung Cell Organotypic Cultures

Pregnant rats were killed by an excess of diethyl ether on day 19 of gestation (term = 22 days). Organotypic cultures of fetal lung cells were established as described previously(7, 10) . The mechanical strain apparatus used in these studies has been described in detail elsewhere (7, 11) . After inoculation, fetal rat lung cells were cultured on Gelfoam sponges in Eagle's minimal essential medium + 10% (v/v) fetal bovine serum. After 24 h, cells were rinsed three times with Eagle's minimal essential medium and cultured in serum-free Eagle's minimal essential medium for another 24 h. Sponges were then subjected to a 5% elongation from their original length, at 60 cycles/min for 15 min/h, which optimally enhanced DNA synthesis and cell division without cell injury(7) . In some experiments, cells were incubated for 3 h with cytochalasin B (1 µM) or herbimycin A (1 µg/ml) prior to the onset of strain.

Cell Lysate Preparation

For most experiments, cells were lysed by placing sponges in radioimmune precipitation assay buffer (1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 158 mM NaCl, 10 mM Tris, 1 mM EGTA, 250 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 1.0 mM benzamidine, 10 mM Na(3)VO(4), and 100 kallikrein inactivator units/ml aprotinin, pH 7.2) for 1 h. Sponges and cells were homogenized using a tissue homogenizer and centrifuged at 12,000 times g for 10 min at 4 °C to remove insoluble debris. Supernatants were stored as aliquots at -70 °C until analyzed.

Triton-soluble and cytoskeletal fractions were prepared according to a modified method of Clark and Brugge(12) . Briefly, sponges were immersed into Triton buffer (2% (v/v) Triton X-100, 2 mM EGTA, 100 mM Tris, 500 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 mM Na(3)VO(4), and 200 kallikrein inactivator units/ml aprotinin, pH 7.2) and incubated overnight at 4 °C. The Triton-soluble fraction was separated by centrifugation of sponges over glass-wool filters. The sponges were then immersed in radioimmune precipitation assay buffer for 4 h. The Triton-insoluble/radioimmune precipitation assay buffer-soluble lysate (cytoskeletal fraction) was collected by centrifugation at 300 times g for 1 min through glass-wool filters. Both fractions were stored as aliquots at -70 °C until analyzed.

Immunoprecipitation and Western Blotting

For immunoprecipitation, cell lysates were adjusted to an equal amount of protein (400-1000 µg) and equal volume, designated antibody (IgG) was added, and samples were incubated at 4 °C overnight. Immune complexes were recovered by the addition of 50 µl of Zysorbin (10% (w/v) Formalin-fixed Staphylococcus aureus Cowan stain A in phosphate-buffered saline) for polyclonal antibodies or 100 µl of protein G-Sepharose (10%, w/v) for monoclonal antibodies. Samples were incubated for 1 h with gentle agitation at 4 °C. The immunoprecipitates were washed twice with lysis buffer. The immunoprecipitated proteins were eluted by boiling in sample buffer (60 mM Tris-HCl, 2% (w/v) SDS, and 5% (v/v) glycerol, pH 6.8) and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes. Nonspecific binding was blocked by incubation with either 3% (w/v) nonfat milk in phosphate-buffered saline or 5% (w/v) bovine serum albumin for 60 min. Membranes were immunoblotted with designated antibody. After a 24-h incubation at 4 °C, the membrane was washed with phosphate-buffered saline and incubated for 60 min at 4 °C with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. After washing, blots were developed with an enhanced chemiluminescence detection kit from Amersham (Oakville, Ontario). Some proteins were detected using I-labeled protein A and autoradiography. For direct Western analyses, cell lysates containing 30 µg of protein were boiled in SDS sample buffer, subjected to SDS-PAGE, and immunoblotted with designated antibodies as described above.

Protein-tyrosine Kinase Measurements

For measuring total PTK activity, sponges were immersed in 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl(2), 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 0.2 mM Na(3)VO(4), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5. Cells were homogenized and centrifuged at 12,000 times g for 2 min at 4 °C, and protein content in the supernatants was determined. An aliquot of the supernatant (10 µl) was incubated at 30 °C in 30 mM HEPES, 10 mM MgCl(2), 0.1 mM dithiothreitol, 20 µM EDTA, 25 µg/ml bovine serum albumin, 0.15% (v/v) Nonidet P-40, 70 µM Na(3)VO(4), 60 µM ATP, and 1 µCi of [-P]ATP, pH 7.4 (total volume = 20 µl), in the presence or absence of a synthetic peptide substrate, RR-SRC (0.5 mM). After a 30-min incubation, the reaction was stopped by adding 20 µl of ice-cold 10% (w/v) trichloroacetic acid. Samples were centrifuged at 12,000 times g for 10 min at 4 °C. Twenty µl of supernatant was spotted on phosphocellulose filters. The filters were washed with 1% (v/v) acetic acid and water and transferred to scintillation vials, and radioactivity was measured. PTK activity was defined as the activity measured in the presence of RR-SRC minus the activity measured in the absence of RR-SRC.

For measurement of Src family PTK activity, a synthetic peptide, [Lys]Cdc2-(6-20)-NH(2), a specific and efficient substrate of Src family PTKs(13) , was used. Two pseudosubstrate peptides, [Val,Ser^14,Lys]Cdc2-(6-20)-NH(2) and [Phe,Lys]Cdc2-(6-20)-NH(2), were used as negative controls. An aliquot (5 µl) of either substrate peptide solution (1.5 mM) or water was mixed with 5 µl of assay buffer (250 mM Tris, 125 mM MgCl(2), 25 mM MnCl(2), and 0.25 mM Na(3)VO(4), pH 7.0) and 10 µl of lysates of either the Triton-soluble or cytoskeletal fraction. The reaction was started by the addition of 5 µl of ATP (0.5 mM) containing 1000 cpm/pmol [-P]ATP at timed intervals and incubated at 30 °C for 30 min. The reaction was stopped by the addition of 10 µl of 50% (w/v) acetic acid and centrifugation at 3000 times g for 5 min. The supernatant (25 µl) was spotted on phosphocellulose filters. The filters were washed four times with 0.75% (v/v) phosphoric acid and once with excess acetone. After drying, filters were transferred to scintillation vials, and radioactivity was measured. PTK activity was defined as the activity measured in the presence of the substrate peptide minus the activity measured in the absence of the peptide. To specifically measure pp60 activity, pp60 was immunoprecipitated from Triton-soluble and cytoskeletal fractions using polyclonal anti-pp60 antibody. The immunoprecipitates were washed twice with dilution buffer (200 mM HEPES, 10% (v/v) glycerol, and 0.1% (v/v) Nonidet P-40, pH 7.0) and resuspended in dilution buffer, and an aliquot of the suspension (10 µl) was then used for the activity assay.

Statistical Analysis

All values are shown as the mean ± S.E. Statistical analysis was by Student's t test or, for comparison of more than two groups, by one-way analysis of variance followed by Duncan's multiple range comparison test with significance defined as p < 0.05.


RESULTS

Strain-induced PTK Activation and Protein Tyrosine Phosphorylation

We have previously shown that mechanical strain-induced fetal lung cell proliferation is mainly mediated through the PLC--PKC pathway(9) . The observed strain-induced PLC-1 tyrosine phosphorylation implies activation of PTKs by mechanical strain. To confirm strain-induced PTK activity, we first measured PTK activity in cell lysates after a short period of strain, using the RR-SRC peptide as a substrate(14) . Total PTK activity increased 10-fold within 15 min of strain, declined slightly during a 45-min resting period, and then increased again during the second period of strain (Fig. 1). We then examined protein tyrosine phosphorylation. Cell lysates were collected after 5, 10, and 15 min of strain; separated by SDS-PAGE; and immunoblotted with antibodies to phosphotyrosine. Increased protein tyrosine phosphorylation occurred 5 min after the onset of strain (Fig. 2A). Specifically, proteins ranging from 110 to 130 kDa were tyrosine-phosphorylated (2-4-fold increase in phosphorylation; p < 0.05) (Fig. 2B), which is a common phenomenon in extracellular matrix-integrin-cytoskeleton-mediated signal transduction(15, 16) .


Figure 1: Mechanical strain induces protein-tyrosine kinase activation. Fetal rat lung cells were subjected to intermittent strain for various times (strain, 0-15 min; relaxation, 15-60 min; strain, 60-75 min) (strain periods are indicated by dashed lines) and then lysed, and total PTK activity was measured by phosphorylation of a PTK-specific peptide substrate, RR-SRC. Data are from a representative experiment (mean ± S.E. from three sponges). Similar results were confirmed in a separate experiment. Statistical analysis was by one-way analysis of variance followed by Duncan's multiple range test. *, p < 0.05 compared with static control.




Figure 2: Mechanical strain induces protein tyrosine phosphorylation. Fetal rat lung cells were subjected to strain for 5-15 min and then lysed, subjected to SDS-PAGE, and analyzed with antibodies to phosphotyrosine. An illustrative blot is shown in A, and results (mean ± S.E.) of densitometric analyses of four separate experiments are shown in B. Statistical analysis was by one-way analysis of variance followed by Duncan's multiple range test. *, p < 0.05 compared with static control.



Mechanical Strain Selectively Increases Tyrosine Phosphorylation of pp60 Substrates

The apparent molecular masses of several characterized pp60 substrates are in the range of 110-130 kDa, e.g. AFAP-110, pp120, pp125, and pp130(17) . To identify the strain-induced tyrosine-phosphorylated proteins, fetal lung cells were subjected to 5 min of strain and lysed, and lysates were immunoprecipitated with polyclonal anti-phosphotyrosine antibody. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with specific antibodies against individual proteins. Alternatively, cell lysates were immunoprecipitated with monoclonal anti-pp125 antibody, which has been shown to immunoprecipitate pp125 from rat embryo fibroblasts(18) , and immunoblotted with anti-phosphotyrosine antibody. The tyrosine phosphorylation of these proteins was compared with that of static controls. Again, 5 min after the onset of strain, tyrosine phosphorylation of proteins corresponding to 110-130 kDa was increased (Fig. 3). The tyrosine phosphorylation of pp120, which was almost undetectable in static culture, was markedly increased by mechanical strain (Fig. 3). The tyrosine phosphorylation of AFAP-110 (2.5-fold increase; n = 3, p < 0.05) and cortactin (pp80/85) was also increased by mechanical strain (Fig. 3). The latter has been identified together with AFAP-110, pp120, pp130, and pp125 to be a substrate for pp60(19) . In contrast, the tyrosine phosphorylation of pp130 and pp125 was not influenced by mechanical strain (Fig. 3). No tyrosine phosphorylation of RasGAP, a protein of 120 kDa implicated in growth factor receptor signaling, was observed in either strained or static cell cultures (data not shown).


Figure 3: Mechanical strain induces tyrosine phosphorylation of pp60 substrates. Fetal rat lung cells were subjected to strain or static culture for 5 min and then lysed. Aliquots of lysates, normalized for protein content, were immunoprecipitated (ptp.) with antibodies to phosphotyrosine (p-tyr) or pp125. Lysates and immunoprecipitates were analyzed by SDS-PAGE. The blots were immunoblotted (blot) as indicated with antibodies to phosphotyrosine, pp120, pp110 (AFAP-110), pp130, and cortactin. Molecular mass marker positions are indicated. Similar results were obtained in two to four separate experiments. C, control static cultures; S, strained cultures.



Strain-induced pp60 Activation and Translocation

pp60 can bind to membrane by lipid anchors and is then well positioned to interact with the membrane-cytoskeleton complex(20) . These features and the observed selective increase in tyrosine phosphorylation of pp60 substrates suggested that pp60 may be activated by mechanical strain. The activities of Src family PTKs in the Triton-soluble and cytoskeletal fractions were measured with a synthetic peptide, [Lys]Cdc2-(6-20)-NH(2). Its relative phosphorylation rates with pp60-related PTKs are up to 180-fold greater than those with non-Src-related PTKs(13) . Total Src PTK activity increased from 8.9 ± 3.2 to 70.4 ± 17.2 pmol/min/mg of protein in the Triton-soluble fraction and from 58.9 ± 17.7 to 117.3 ± 60.4 pmol/min/mg of protein in the cytoskeletal fraction. No phosphorylation of two pseudosubstrates (see ``Experimental Procedures'') was observed (data not shown). To test the specific activity of pp60, pp60 in the Triton-soluble and cytoskeletal fractions was immunoprecipitated with polyclonal anti-pp60 antibody. The PTK activities in the immunoprecipitates were then determined using the [Lys]Cdc2-(6-20)-NH(2) peptide as substrate. The specific activity of pp60 in the Triton-soluble fraction did not change, while that in the cytoskeletal fraction increased 3.77 ± 0.73-fold (p < 0.05) after 5 min of mechanical strain (Fig. 4). The percentage of measurable pp60 activity in the Triton-soluble fraction decreased, whereas a corresponding increase of pp60 activity in the cytoskeletal fraction (from 11 to 35%) was noted. The subcellular distribution of pp60 was also determined by Western blotting using anti-pp60 mAb. In static culture, pp60 was mainly present in the Triton-soluble fraction (Fig. 5). The amount of pp60 in the cytoskeletal fraction increased after 5 min of strain (Fig. 5). When cells were preincubated with cytochalasin B (1 µM) to interrupt the dynamic polymerization of actin filaments, mechanical strain did not induce pp60 activation and translocation to the cytoskeletal fraction ( Fig. 4and Fig. 5). Preincubation of cells with herbimycin A (1 µg/ml) inhibited both basal and strain-induced pp60 activities (Fig. 4) and blocked strain-induced pp60 translocation to the cytoskeletal fraction (Fig. 5). These results suggest that pp60 activity in fetal lung cells depends on its association with the cytoskeletal matrix and that mechanical strain-induced translocation of pp60 requires the integrity of actin filaments as well as the existence of tyrosine-phosphorylated proteins prior to the onset of strain. The tyrosine phosphorylation of pp60 was determined by immunoprecipitation of Triton-soluble and cytoskeletal fractions with polyclonal anti-phosphotyrosine antibody followed by immunoblotting with anti-pp60 mAb. No significant difference between control and strained cells was observed (Fig. 6), which may be due to the simultaneous dephosphorylation of tyrosine-phosphorylated Tyr(P)-527 and phosphorylation of Tyr-416 (21) .


Figure 4: Mechanical strain induces pp60activation in the cytoskeleton. Fetal rat lung cells were preincubated for 3 h with or without either 1 µM cytochalasin B or 1 µg/ml herbimycin A and then subjected to strain or static culture for 5 min. Triton-soluble and cytoskeletal fractions were prepared, and aliquots, equalized for protein content, were immunoprecipitated with polyclonal antibodies to pp60. PTK activity in the immunoprecipitates was assayed using peptide [Lys]Cdc2-(6-20)-NH(2) as substrate and is expressed as pmol/min/mg of protein. Data are mean ± S.E. from three sponges. A, Triton-soluble fraction; B, cytoskeletal fraction. Non, untreated cells; Cyto.B, cytochalasin B; Herb.A, herbimycin A. *, p < 0.05 compared with static controls.




Figure 5: Mechanical strain induces pp60translocation to the cytoskeleton. Fetal rat lung cells were preincubated for 3 h with or without either 1 µM cytochalasin B or 1 µg/ml herbimycin A and then subjected to strain or static culture for 5 min. Triton-soluble (sol) and cytoskeletal (cysk) fractions were prepared, and aliquots, equalized for protein content, were immunoblotted with antibodies to pp60 (mAb GD11). The position of pp60 is indicated. Similar results were obtained in two separate experiments. C, control static cultures; S, strained cultures.




Figure 6: Mechanical strain does not influence pp60 tyrosine phosphorylation. Fetal rat lung cells were subjected to strain or static culture for 5 min. Triton-soluble (sol) and cytoskeletal (cysk) fractions were prepared, and aliquots, equalized for protein content, were immunoprecipitated (i.p.) with antibodies to phosphotyrosine (p-tyr) and immunoblotted (blot) with antibodies to pp60 (mAb GD11). A representative blot is shown in A, and the mean and the range of densitometric analysis of blots from two separate experiments are shown in B.



Mechanical Strain-induced pp60 Translocation to Cytoskeleton Is Mediated through AFAP-110

We further examined the mechanism(s) by which pp60 is translocated to the cytoskeleton. Several pp60 substrates are either cytoskeletal proteins or associated with the cytoskeleton. These include pp125(22) ; pp120, a tyrosine-phosphorylated protein that shares sequence similarity with cadherin-binding factors (23) ; AFAP-110(24, 25) ; cortactin, an 80/85-kDa filamentous actin-binding protein that is enriched in the cell cortex(19) ; and paxillin, a focal adhesion protein associated with vinculin(26) . Both pp120 and cortactin were coimmunoprecipitable with pp60, but the association was not affected by mechanical strain (Fig. 7). Paxillin was tyrosine-phosphorylated (data not shown) and coimmunoprecipitated with pp60 (Fig. 8). However, these characteristics were not altered by mechanical strain. No association between pp60 and pp125 or pp130 was observed (Fig. 8). In contrast, the association between pp60 and AFAP-110 increased 3-fold after a 5-min strain period (p < 0.05, n = 3) (Fig. 7). These results suggest that the strain-induced pp60 translocation to the cytoskeletal matrix is, at least partially, mediated through this actin filament-associated protein.


Figure 7: Mechanical strain induces association of pp60 with AFAP-110. Fetal rat lung cells were subjected to strain for 5 min and then lysed. Cell lysates, normalized for protein content, were immunoprecipitated (ptp.) with polyclonal anti-pp60 antibody. Lysates(-) and immunoprecipitates (Src) were analyzed by SDS-PAGE. The blots were immunoblotted (blot) as indicated with antibodies to pp120, cortactin, and pp110 (AFAP-110). Positions are indicated by arrowheads. Similar results were obtained in two to four separate experiments. C, control static cultures; S, strained cultures.




Figure 8: Mechanical strain does not activate the pp125-paxillin pathway. Fetal rat lung cells were subjected to strain for 5 min and then lysed. Cell lysates, equalized for protein content, were immunoprecipitated (ptp.) with polyclonal anti-pp60 or monoclonal anti-pp125 antibody. Lysates(-) and immunoprecipitates (Src or FAK) were analyzed by SDS-PAGE. The blots were immunoblotted (blot) as indicated with antibodies to pp130, paxillin, and Src. Positions are indicated by arrowheads. Similar results were obtained in two separate experiments. C, control static cultures; S, strained cultures.



PTK Activation Mediates PLC-1 Activation

To determine whether PTK activation is related to the strain-induced PLC--PKC activation, cells were preincubated with or without herbimycin A (1 µg/ml), a PTK inhibitor, and then subjected to static culture or mechanical strain. Consistent with our previous observation(9) , tyrosine phosphorylation of PLC-1 was increased after 5 min of strain (Fig. 9, left panel). Both basal and strain-induced PLC-1 tyrosine phosphorylations were blocked by herbimycin A. When cell lysates were immunoprecipitated with anti-pp60 mAb, electrophoresed, and immunoblotted with anti-PLC-1 mAb, an association between pp60 and PLC-1 was observed in fetal rat lung cells. The association was significantly increased by mechanical strain (p < 0.05) and attenuated by pretreatment with herbimycin A (Fig. 9, right panel). Herbimycin A also completely blocked the enhanced DNA synthesis induced by a 48-h intermittent mechanical strain regimen (Fig. 10).


Figure 9: Strain-induced PLC-1 tyrosine phosphorylation and PLC-1-pp60 association are blocked by the PTK inhibitor herbimycin A. Fetal rat lung cells were preincubated for 3 h with or without 1 µg/ml herbimycin A and then subjected to strain or static culture. Cells were lysed after 5 min of strain, and cell lysates, normalized for protein content, were immunoprecipitated (ptp.) with antibodies to phosphotyrosine (p-tyr; left panel) or pp60(right panel). The immunoprecipitates were electrophoresed and immunoblotted (blot) with antibodies to PLC-1. The position of PLC-1 is indicated. Similar results were obtained in two separate experiments. C, control static cultures; S, strained cultures.




Figure 10: Strain-induced DNA synthesis is blocked by the PTK inhibitor herbimycin A. Fetal rat lung cells were preincubated with or without 1 µg/ml herbimycin A for 3 h and then subjected to a 48-h intermittent strain or static culture. [^3H]Thymidine incorporation into DNA was measured. Data are mean ± S.E. from three sponges. Statistical analysis was by one-way analysis of variance followed by Duncan's multiple range test. *, p < 0.05 compared with the static control group.




DISCUSSION

Mechanical strain-induced increases of inositol 1,4,5-trisphosphate and diacylglycerol have been observed in various cell types, such as endothelial cells(27) , vascular smooth muscle cells(28) , cardiac myocytes(29) , skeletal muscle(30) , and bone cells (31) as well as fetal lung cells(9) . The increase of inositol 1,4,5-trisphosphate and diacylglycerol is followed by PKC activation, and blockage of PLC or PKC activity can inhibit physical force-induced cell proliferation(9, 28) . Therefore, PLC-PKC seems to be a common pathway by which cells transduce physical signals. How this pathway becomes activated is unknown. In this study, mechanical strain of fetal lung cells rapidly activated PTKs and increased tyrosine phosphorylation of several phosphotyrosine-containing proteins, AFAP-110, pp120, and cortactin. The phosphorylation of these pp60 substrates by mechanical strain coincided with pp60 activation and translocation to the cytoskeletal compartment. The PTK inhibitor herbimycin A blocked strain-induced PLC-1 tyrosine phosphorylation, PKC activation, and DNA synthesis, suggesting that the rapid activation of cytoplasmic PTKs is an upstream event of the PLC--PKC pathway. In vitro studies have shown that PLC-1 can be tyrosine-phosphorylated by pp60 and other Src family PTKs(32) . In platelets, electrotransjection of monoclonal pp60 antibody inhibited activation of PLC-1(33) . Moreover, pp60 has been coimmunoprecipitated with PLC-1 in platelets(33) , and this association was increased after treatment of platelets with platelet-activating factor(33) . Similarly, we observed coimmunoprecipitation of pp60 and PLC-1 in fetal rat lung cells, and this association was increased by mechanical strain and inhibited by herbimycin A treatment. Thus, our present data are compatible with strain-induced PLC-1 tyrosine phosphorylation in fetal lung cells being, at least in part, mediated via pp60. Phosphorylated PLC- then activates the PKC pathway and downstream signal cascades.

Increased tyrosine phosphorylation of proteins ranging from 110 to 130 kDa is a common phenomenon in extracellular matrix-integrin-cytoskeleton-mediated signal transduction initiated by integrin clustering (15) or cell attachment(16) . An integrin-related increase of tyrosine phosphorylation has been found for proteins such as pp130 (15) and pp125(16, 34) . Although tyrosine phosphorylation of pp130 and pp125 was observed in fetal lung cells, it was not affected by short periods of mechanical strain, and the association between these two proteins and pp60 in fetal rat lung cells was not detected by coimmunoprecipitation. Paxillin has been suggested to be a direct substrate for pp125(35) and has been found to bind in vitro to the SH3 domain of c-Src(36) . In this study, coimmunoprecipitation of pp60 and paxillin was demonstrated in fetal rat lung cells. However, the tyrosine phosphorylation and association of paxillin with pp60 were not altered by mechanical strain. The strain-induced pp60 translocation therefore appears not to be mediated through the pp125-paxillin pathway. In addition, mechanical strain-induced cell proliferation was not affected when fetal lung cells were preincubated with an RGD peptide to block the extracellular matrix-integrin interaction. (^2)Taken together, these results suggest that the mechanical strain-induced signals are different from those initiated by integrins, although the cytoskeleton system appears to be involved in the transmission of both signals.

Tyrosine phosphorylation of pp130, pp125, pp120, and cortactin has been observed upon epidermal growth factor, PDGF, and colony-stimulating factor-1 stimulation of several cell types(35, 37, 38) . The effect of these growth factors is likely mediated through intermediate PTKs such as Src family members. PDGF stimulation of quiescent NIH 3T3 cells and human fibroblasts activates Src family tyrosine kinases(39) . Treatment of A172 glioblastoma cells with PDGF or epidermal growth factor induces activation and translocation of c-Src to the cytoskeleton(40) . We have recently demonstrated that strain-enhanced proliferation is mediated via PDGF(8) . However, it is unlikely that strain-induced pp60 activation is a result of an exocytosis of PDGF. We found no measurable changes of PDGF in the culture medium within 4 h of mechanical strain(8) . Tyrosine phosphorylation of RasGAP has been observed for various cell types upon activation of epidermal growth factor or PDGF receptors(35, 41, 42) . We also observed tyrosine phosphorylation of RasGAP and PDGF beta-receptors after PDGF-BB treatment of fetal rat lung cells. In contrast, we did not observe tyrosine phosphorylation of RasGAP or PDGF beta-receptors after 5 min of mechanical strain (data not shown).

One of the regulatory mechanisms for pp60 activation is through its association with the cytoskeletal matrix(43) . All oncogenic variants of pp60 have been shown to be tightly associated with the Triton-insoluble cytoskeletal matrix, and this association correlates with the elevated tyrosine kinase activity of pp60(44, 45, 46) . Normally, pp60 does not associate with the detergent-insoluble cellular matrix(44) . However, redistribution of activated pp60 to the cytoskeletal matrix has been reported for thrombin-stimulated platelets (12) and growth factor-stimulated A172 glioblastoma cells(40) . The reason for Src activation following its translocation to the cytoskeleton is unknown. In the current model for Src regulation, the tyrosine-phosphorylated C-terminal Tyr(P)-527 sequence binds by an intramolecular interaction to Src's own SH2 domain to maintain Src in an inactive state(47, 48) . The tyrosine-phosphorylated Src C-terminal Tyr(P)-527 sequence does, however, not resemble the consensus high affinity SH2-binding site and therefore binds poorly to the SH2 domain of Src. In contrast, AFAP-110, a distinctive cytoskeleton-associated protein(24, 25) , contains four putative SH2-binding sites including a consensus high affinity tyrosine-phosphorylated SH2-binding site(25) . Mechanical strain-induced cytoskeleton deformation may physically facilitate the approximation of these binding sites to pp60 and activate pp60 by competing with Src Tyr(P)-527 for Src SH2 binding. In this study, we indeed observed an increased association between pp60 and AFAP-110. The increased association between AFAP-110 and pp60 may activate pp60, which results in an increased tyrosine phosphorylation of AFAP-110 and other proteins, such as PLC-1.


FOOTNOTES

*
This work was supported by a group grant (to M. P. and A. K. T.) and Operating Grant MT-13270 (to M. L.) from the Medical Research Council of Canada, by Grant R01HL43416 from the National Institutes of Health (to M. P.), by an operating grant (to M. L.) and equipment grants (to M. P., A. K. T., and M. L.) from the Ontario Thoracic Society, and by the Dean's Fund from the Faculty of Medicine, University of Toronto (to M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Neonatology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6773; Fax: 416-813-5002.

(^1)
The abbreviations used are: PLC-1, phospholipase C-1; PKC, protein kinase C; AFAP-110, actin filament-associated protein of 110 kDa; PTK, protein-tyrosine kinase; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PDGF, platelet-derived growth factor.

(^2)
M. Liu and M. Post, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. J. T. Parsons for the gifts of anti-AFAP-110 and anti-pp130 antibodies.


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