Reactive oxidant and p42/44 MAP kinase signaling is necessary for mechanical strain-induced proliferation in pulmonary epithelial cells.

Mechanical strain is necessary for normal lung growth and development. Individuals with respiratory failure are supported with mechanical ventilation, leading to altered lung growth and injury. Understanding signaling pathways initiated by mechanical strain in lung epithelial cells will help guide development of strategies aimed at optimizing strain-induced lung growth while mitigating ventilator-induced lung injury. To study strain-induced proliferative signaling, focusing on the role of reactive oxidant species (ROS) and p42/44 mitogen-activated protein (MAP) kinase, human pulmonary epithelial H441 and MLE15 cells were exposed to equibiaxial cyclic mechanical strain. ROS were increased within 15 min of strain. N-acetylcysteine inactivated strain-induced ROS and inhibited p42/44 MAP kinase phosphorylation and strain-induced proliferation. PD98059 and UO126, p42/44 MAP kinase inhibitors, blocked strain-induced proliferation. To verify the specificity of p42/44 MAP kinase inhibition, cells were transfected with dominant-negative mitogen-activated protein kinase kinase-1 plasmid DNA. Transfected cells did not proliferate in response to mechanical strain. To determine whether strain-induced tyrosine kinase activity is necessary for strain-induced ROS-p42/44 MAP kinase signaling, genistein, a tyrosine kinase inhibitor, was used. Genistein did not block strain-induced ROS production or p42/44 MAP kinase phosphorylation. Gadolinium, a mechanosensitive calcium channel blocker, blocked strain-induced ROS production and p42/44 MAP kinase phosphorylation but not strain-induced tyrosine phosphorylation. These data support ROS production and p42/44 MAP kinase phosphorylation being involved in a common strain-induced signaling pathway, necessary for strain-induced proliferation in pulmonary epithelial cells, with a parallel strain-induced tyrosine kinase pathway.     

Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells

Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular signal-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or focal adhesion kinase (FAK) mediates cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/min. Cyclic strain activated Src within 2 min by increasing phosphorylation at Tyr⁴¹⁸, followed by rapid phosphorylation of FAK at Tyr³⁹⁷ and Tyr⁵⁷⁶ and ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (n = 5, P < 0.05). Twenty-four (A549 cells) and 24–72 h (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty-four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 µmol/l PP2 or short interfering RNA targeted to Src) or MEK (50 µmol/l PD-98059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr⁵⁷⁶ and ERK-Thr²⁰²/Tyr²⁰⁴ but not FAK-Tyr³⁹⁷. Reducing FAK by FAK-targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain-induced ERK activation but not strain-induced Src phosphorylation. Together, these results suggest that repetitive mechanical deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling. extracellular signal-regulated kinase; mitogenic; signaling     

Mechanical Strain Increases Protein Tyrosine Phosphorylation in Airway Smooth Muscle Cells

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflectedin vitrowhere cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125^FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125^FAKand paxillin. Tyrosine phosphorylation decreased by 4 h in pp125^FAKafter discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.     

Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or G?-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.     

Induction of interleukin (IL)-1α and β gene expression in human keratinocytes exposed to repetitive strain: Their role in strain-induced keratinocyte proliferation and morphological change

Recent studies in our laboratory have demonstrated that mechanical strain alters many facets of keratinocyte biology including proliferation, protein synthesis, and morphology. IL-1 is known to play an important role in the autocrine regulation of these basic cellular properties under basal and stimulated conditions. However, it is not known whether IL-1 plays a role in strain-induced alteration of keratinocyte biology. Thus, the objective of this study was to test the hypothesis that cyclic strain stimulates IL-1 expression and that strain-induced changes in keratinocyte function is regulated by IL-1. To test this hypothesis, we examined the effect of cyclic strain (10% average deformation) on keratinocyte IL-1 gene expression and the effect of neutralizing antibodies of IL-1α and IL-1β on strain-induced changes in keratinocyte proliferation, morphology, and orientation. Northern blot analyses demonstrated that steady state levels of IL-1α and β mRNA were elevated by 4 h, peaked at 12 h of cyclic strain (IL-1α, 304 ± 14.2%; IL-1β, 212 ± 5.6% increase vs. static controls) and decreased gradually by 24 h. IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.01 μg/ml) significantly blocked strain-induced keratinocyte proliferation as well as the basal rate of proliferation. In contrast, IL-1 antibodies (IL-1α, 0.01 μg/ml; IL-1β, 0.1 μg/ml) had no effect on strain-induced morphological changes such as elongation and alignment. We conclude that mechanical strain induces IL-1 mRNA expression in keratinocytes. The role of IL-1 in mediating strain-induced changes in keratinocyte biology remains to be determined but appears to be independent of morphological changes. J. Cell. Biochem. 69:95–103, 1998. © 1998 Wiley-Liss, Inc.     

17beta -Estradiol modulates mechanical strain-induced MAPK activation in mesangial cells.

Gender is an important determinant of clinical outcome across a broad spectrum of kidney diseases, but the mechanism(s) responsible for the protective effect of female gender have not been fully elucidated. Remnant kidney glomerular injury is limited in female rats compared with male rats despite similar elevations in glomerular capillary pressure. In vitro, mechanical strain leads to the activation of p44/42 mitogen-activated kinase (p44/42 MAPK) and Jun N-terminal kinase/stress-activated protein kinase (SAPK) in glomerular mesangial cells (MC). Accordingly, we studied the effect of 17beta-estradiol on mechanical strain-induced signal transduction in MC. Exposure of MC to mechanical strain increased p44/42 MAPK activation (3-fold) and SAPK activation (2.5-fold), and kinase activation was inhibited by pretreatment with 17beta-estradiol (10(minus sign8) to 10(minus sign11) m) for 24 h in a dose-dependent manner. Mechanical strain-induced nuclear translocation of p44/42 MAPK and SAPK and nuclear protein binding to AP-1 were also attenuated by 17beta-estradiol. The inhibitory effects of 17beta-estradiol were not reproduced by the cell-impermeable estrogen, BSA/17beta-estradiol, nor did preincubation with 17beta-estradiol lead to actin cytoskeleton disassembly or impaired stress fiber formation. However, 17beta-estradiol did increase base-line levels of the dual specificity phosphatase MKP-1. The inhibitory effects of 17beta-estradiol on p44/42 MAPK activation and SAPK activation, translocation, and AP-1 binding were all abrogated by the estrogen receptor antagonist, ICI-182,780. We conclude that attenuation of mechanical strain-induced MAPK activation by 17beta-estradiol is dependent on intracellular estrogen receptor. The attenuation of stretch-induced kinase activation may be due, at least in part, to an effect of 17beta-estradiol on MKP-1 expression. Together, these findings add insight into the protective effect of gender on renal disease progression.     

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.     

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor beta

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

NO inhibits stretch-induced MAPK activity by cytoskeletal disruption

Mesangial cells (MC) grown on extracellular matrix protein-coated plates and exposed to cyclic strain/relaxation proliferate and produce extracellular matrix protein, providing an in vitro model of signaling in stretched MC. Intracellular transduction of mechanical strain involves mitogen-activated protein kinases, and we have shown that p42/44 mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) is activated by cyclic strain in MC. In vivo studies show that increased production of nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing glomerular capillary pressure, and we have observed that NO attenuates stretch-induced ERK activity in MC via generation of cyclic guanosine monophosphate (cGMP). Accordingly, we sought to determine whether NO affects strain-induced ERK activity after strain and how this is mediated. Strain-induced ERK activity was dependent on time and magnitude of stretch and was maximal after 10 min at -27 kilopascals. Actin cytoskeleton disruption with cytochalasin D abrogated this. The non-metabolizable cGMP analogue 8-bromo cyclic GMP (8-Br-cGMP) dose-dependently attenuated strain-induced ERK activity. Cytoskeletal stabilization with jasplakinolide prevented this inhibitory effect of 8-Br-cGMP. Cyclic strain increased nuclear translocation of phospho-ERK by immunofluorescent microscopy, again attenuated by 8-Br-cGMP. Jasplakinolide prevented the inhibitory effect of 8-Br-cGMP on activated ERK nuclear translocation after strain. Strain increased ERK-dependent AP-1 nuclear protein binding, which was attenuated by cytochalasin D and 8-Br-cGMP. These data indicate that cGMP can inhibit cyclic strain-induced ERK activity, nuclear translocation, and AP-1 nuclear protein binding. Cytoskeletal disruption leads to the same effect, whereas cytoskeleton stabilization reverses the effect of 8-Br-cGMP. Thus, NO inhibits strain-induced ERK activity by cytoskeletal destabilization.     

ILK mediates the effects of strain on intestinal epithelial wound closure

The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.     

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: Study of strain dependency and the role of protein kinase A and C signaling pathways

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0–7% center; 7–24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7–24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0–7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 μM Rp-cAMP) or PKC (with 5–20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses. J. Cell. Physiol. 170:228–234, 1997. © 1997 Wiley-Liss, Inc.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

Mechanical strain increases PDGF-B and PDGF beta receptor expression in vascular smooth muscle cells

Cyclic mechanical strain causes proliferation of vascular smooth muscle cells, mediated in part by platelet-derived growth factor (PDGF). We examined the effect of cyclic strain on expression of PDGF-B and the PDGF beta receptor. Neonatal rat vascular smooth muscle cells were exposed to 1 hertz cyclic strain on silicone elastomer plates. PDGF-B mRNA increased after 6 h of strain. In cells transfected with a PDGF-B promoter chloramphenicol acetyl transferase construct (psisCAT 6A), activity increased by 12-fold following 12 h of strain. Two neutralizing antibodies to the PDGF beta receptor both reduced strain-induced [(3)H]thymidine incorporation by 50%. Expression of the PDGF beta receptor protein increased 1.8-fold following 24 h of strain. During strain, PDGF beta receptor expression was not significantly altered by neutralizing antibodies to PDGF-B. Thus, both PDGF-B and the PDGF beta receptor are induced by cyclic mechanical strain and both contribute to cell proliferation in response to strain.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: an in vitro model for distraction osteogenesis

Factors known to regulate bone production during distraction osteogenesis include mechanical strain on bone forming cells and up-regulation of transforming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was used to evaluate the functional effect of exogenous TGF-beta1 on mitogenesis in murine-derived MC3T3 osteoblasts during the period of active mechanical strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteoblasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 24 hours, all cells were transferred to media containing TGF-beta1, and strain was continued for an additional 48 hours. The study was repeated by using two doses of TGF-beta1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta1 in both the nonstrained and strained groups (p < 0.0001). The final cell count in the strained group was significantly less than that in the nonstrained group (p < 0.0001) for both concentrations of TGF-beta1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the mitogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypothesis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteoblasts. This study demonstrated no significant differences in cell counts after addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates the functional significance of mechanical strain on osteoblast cell counts. Furthermore, this may help to explain the temporal relationship observed during the early distraction (strain) phase of distraction osteogenesis in rodent models in which peak up-regulation of TGF-beta1 gene expression correlates with peak suppression of osteoblast function as measured by gene expression of extracellular matrix proteins.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.