Inhibited Fibroblast Migration Research Articles

Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration.

Epimorphic regeneration proceeds with or without formation of a blastema, as observed for the limb and skin, respectively. Inhibition of epimorphic regeneration provides a means to interrogate the cellular and molecular mechanisms that regulate it. In this study, we show that exposing amputated limbs to beryllium nitrate disrupts blastema formation and causes severe patterning defects in limb regeneration. In contrast, exposing full-thickness skin wounds to beryllium only causes a delay in skin regeneration. By transplanting full-thickness skin from ubiquitous GFP-expressing axolotls to wild-type hosts, we demonstrate that beryllium inhibits fibroblast migration during limb and skin regeneration in vivo Moreover, we show that beryllium also inhibits cell migration in vitro using axolotl and human fibroblasts. Interestingly, beryllium did not act as an immunostimulatory agent as it does in Anurans and mammals, nor did it affect keratinocyte migration, proliferation or re-epithelialization, suggesting that the effect of beryllium is cell type-specific. While we did not detect an increase in cell death during regeneration in response to beryllium, it did disrupt cell proliferation in mesenchymal cells. Taken together, our data show that normal blastema organogenesis cannot occur without timely infiltration of local fibroblasts and highlights the importance of positional information to instruct pattern formation during regeneration. In contrast, non-blastemal-based skin regeneration can occur despite early inhibition of fibroblast migration and cell proliferation.

MCPIP1 Regulates Fibroblast Migration in 3-D Collagen Matrices Downstream of MAP Kinases and NF-κB

The fibroblast-populated 3D collagen matrix has been used to model matrix contraction, cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein-induced protein 1) has been shown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstrated induction of MCPIP1 in human fibroblasts embedded in the stress-released 3D collagen matrix, which occurred through activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore, MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix. MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibition of migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role for MCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model.

Targeting the Chemokines in Cardiac Repair

Chemokines are a family of chemotactic cytokines that play an essential role in leukocyte trafficking. Upregulation of both CC and CXC chemokines is a hallmark of the inflammatory and reparative response following myocardial infarction. Release of danger signals from dying cells and damaged extracellular matrix activates innate immune pathways that stimulate chemokine synthesis. Cytokineand chemokine-driven adhesive interactions between endothelial cells and leukocytes mediate extravasation of immune cells into the infarct. CXC chemokines (such as interleukin-8) are bound to glycosaminoglycans on the endothelial surface and activate captured neutrophils, inducing expression of integrins. CC chemokines (such as monocyte chemoattractant protein (MCP)-1) mediate recruitment of proinflammatory and phagocytotic mononuclear cells into the infarct. CC Chemokines may also regulate late infiltration of the healing infarct with inhibitory leukocytes that suppress inflammation and restrain the post-infarction immune response. Non-hematopoietic cells are also targeted by chemokines; in healing infarcts, the CXC chemokine Interferon-γ inducible Protein (IP)-10 exerts antifibrotic actions, inhibiting fibroblast migration. Another member of the CXC subfamily, Stromal cell-derived Factor (SDF)-1, may protect the infarcted heart by activating pro-survival signaling in cardiomyocytes, while exerting angiogenic actions through chemotaxis of endothelial progenitors. Several members of the chemokine family may be promising therapeutic targets to attenuate adverse remodeling in patients with myocardial infarction.

Salvianolic Acid A, a Novel Matrix Metalloproteinase-9 Inhibitor, Prevents Cardiac Remodeling in Spontaneously Hypertensive Rats

Cardiac fibrosis is a deleterious consequence of hypertension which may further advance to heart failure and increased matrix metalloproteinase-9 (MMP-9) contributes to the underlying mechanism. Therefore, new therapeutic strategies to attenuate the effects of MMP-9 are urgently needed. In the present study, we characterize salvianolic acid A (SalA) as a novel MMP-9 inhibitor at molecular, cellular and animal level. We expressed a truncated form of MMP-9 which contains only the catalytic domain (MMP-9 CD), and used this active protein for enzymatic kinetic analysis and Biacore detection. Data generated from these assays indicated that SalA functioned as the strongest competitive inhibitor of MMP-9 among 7 phenolic acids from Salvia miltiorrhiza. In neonatal cardiac fibroblast, SalA inhibited fibroblast migration, blocked myofibroblast transformation, inhibited secretion of intercellular adhesion molecule (ICAM), interleukin-6 (IL-6) and soluble vascular cell adhesion molecule-1 (sVCAM-1) as well as collagen induced by MMP-9 CD. Functional effects of SalA inhibition on MMP-9 was further confirmed in cultured cardiac H9c2 cell overexpressing MMP-9 in vitro and in heart of spontaneously hypertensive rats (SHR) in vivo. Moreover, SalA treatment in SHR resulted in decreased heart fibrosis and attenuated heart hypertrophy. These results indicated that SalA is a novel inhibitor of MMP-9, thus playing an inhibitory role in hypertensive fibrosis. Further studies to develop SalA and its analogues for their potential clinical application of cardioprotection are warranted.

Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

A Chitosan‐Based Sinus Sealant for Reduction of Adhesion Formation in Rabbit and Sheep Models

Chronic sinusitis is the most prevalent chronic disease in the United States in adults aged 18 to 44 years, with approximately 250,000 operations performed annually. Although often successful, sinus surgery fails in greater than 15% of patients. Adhesion formation is a common complication and cause for subsequent revision surgery. Here, the authors evaluate a sprayable chitosan/starch-based sinus sealant and demonstrate its ability to reduce adhesion formation both in vitro and in 2 animal models. Randomized, controlled, animal trials. Academic medical center (fibroblast experiments) and animal laboratories (sheep and rabbit studies). This sinus sealant was applied to human cultured fibroblasts obtained from surgically removed polyps to examine its ability to inhibit fibroblast migration and proliferation. The sinus sealant was applied to New Zealand White rabbits (n = 20) in an established cecal-sidewall abrasion model and to sheep (n = 10) in a sinus surgical adhesion model to examine its ability to reduce adhesion formation. This sinus sealant inhibited migration and proliferation of human cultured fibroblasts and reduced the total adhesion score from 4.9 to 0.3 for a total reduction of 94% (95th percentile confidence interval [CI], 78%, 100%; P < .001) in a well-established rabbit cecal-sidewall model commonly used for adhesion testing. Moreover, this sealant reduced adhesion formation from 80% to 10% for a total reduction of 70% (95th percentile CI, 57%, 93%; P = .003) in a sheep sinus adhesion surgical model. This chitosan-based sealant demonstrates promise for reducing adhesion formation in sinus surgery.

In Vitro Effects of Pirfenidone on Cardiac Fibroblasts: Proliferation, Myofibroblast Differentiation, Migration and Cytokine Secretion

Cardiac fibroblasts (CFs) are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.

Interaction of TGF-β1 and Ionizing Radiation in an In Vitro Wound Healing Model: Rho Kinase-mediated Inhibition of Fibroblast Migration and Implications for IORT

Interaction of TGF-β1 and Ionizing Radiation in an In Vitro Wound Healing Model: Rho Kinase-mediated Inhibition of Fibroblast Migration and Implications for IORT

Abstract 1400: Inhibition of cell migration by the synthetic triterpenoid acetylenic tricyclic bis-(cyano enone)

Abstract Triterpenoids are a family of naturally occurring compounds that have multiple anti-tumorigenic effects, including the ability to induce cell differentiation, apoptosis and inhibit proliferation. Research in our lab has demonstrated that a synthetically derived 5 ring triterpenoid, 2-cyano-3, 12-dioxooleana-1,9-dien-28-oic acid-imidazolide (CDDO-Im), inhibits cell migration (To et al., 2008, J. Biol. Chem. 283:11700), which is essential in numerous physiological processes including tumor metastasis. Recently, a smaller 3 ring compound containing the same active functional groups as CDDO-Im has been derived; acetylenic tricyclic bis-(cyano enone) [(+/-)-(4aa,8aa,10ab)-1,2,4a,6,8a,9,10,10a-octahydro-8a-ethynyl-1,1,4a-trimethyl-2,6 dioxophenanthrene-3,7-dicarbonitrile] (TBE-31). Here we examined the effect of TBE-31 on cell migration. Using scratch assays with Rat2 fibroblasts we demonstrated that concentrations of TBE-31 above 0.75 µM were effective in inhibiting fibroblast migration. To elucidate the mechanism(s) for this inhibition we examined different components of the cytoskeleton and the polarity complex, which are both crucial for cell migration. To examine cell polarity, we used immunofluorescence microscopy to assess the localization of two proteins involved in the polarization of migrating cells, IQGAP1 and RAC1. Similar to CDDO-Im, TBE-31 was observed to reduce the elongation and polarity of cells, however unlike CDDO-Im, IQGAP1 and RAC1 in TBE-31 treated cells were localized in punctate fashion around the cell perimeter instead of at the leading edge. Using immunofluorescence imaging, the cytoskeleton of Mv1Lu mink lung cells or COS7 cells treated with TBE-31 were analyzed. The microtubule network of treated cells displayed a unique cross-hatched phenotype and were resistant to nocodazole depolymerization. In addition, the intermediate filament cytoskeleton was observed to retract towards the nucleus. Lastly, in vitro actin polymerization assays revealed that TBE-31 inhibits linear but not branched actin polymerization. We conclude that TBE-31 is an effective inhibitor of cell migration and further research will shed light on the mechanisms that TBE-31 uses to modulate the cell migration machinery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1400. doi:10.1158/1538-7445.AM2011-1400

The role of CXCR3 signaling in myocardial infarction

Chemokine signaling regulates the reparative response following myocardial infarction and plays an important role in the pathogenesis of cardiac remodeling. CXCR3, the main functional receptor for IP‐10, Mig and I‐TAC, is markedly induced in healing myocardial infarcts. We have recently demonstrated that IP‐10 critically regulates infarct healing by inhibiting fibroblast migration. Our current investigation tests the hypothesis that IP‐10 actions in the infarcted myocardium may be mediated through CXCR3. Accordingly, we examined the effects of CXCR3 loss on infarct healing and cardiac remodeling. The CXCR3 ligands IP‐10 and MIG but not I‐TAC were upregulated in reperfused mouse infarcts. Flow cytometry analysis of cell suspensions harvested from mouse infarcts after 72h of reperfusion identified large populations of CD3+ T lymphocytes, CD14+ monocytes, CD31+ endothelial cells and alpha‐smooth muscle actin +/collagen I+ myofibroblasts expressing CXCR3. Much like IP‐10 −/− mice, CXCR3 −/− mice exhibited increased infiltration of the infarcted myocardium with neutrophils and macrophages. However, in contrast to the increased infiltration of the infarct border zone with myofibroblasts observed in IP‐10 −/− mice, CXCR3 −/− hearts had reduced and delayed myofibroblast infiltration. In vitro experiments demonstrated that the inhibitory effects of IP‐10 on bFGF ‐induced cardiac fibroblast migration were independent of CXCR3. In contrast to the increased remodeling observed in IP‐10 deficient mice, CXCR3 null mice and WT animals had comparable dilation following infarction. The findings suggest that the inhibitory effects of IP‐10 on fibroblast migration protect from the development of adverse remodeling and are mediated through CXCR3‐independent pathways.

Dermacentor variabalis: Tick saliva inhibits osteosarcoma (Saos) cell migration and invasion

Previous studies from our laboratory show that tick saliva inhibits fibroblast migration in both wound healing and chemotaxis assays. These results lead us to examine the effect of tick saliva on the invasiveness of Saos cells. Assessment of cell migration using a blind well assay showed that saliva decreased basal cell migration by 57.78+5.17% and inhibited stimulated migration by 67.41+8.87%. In addition[j1], saliva reduced SaOS invasiveness by 40% as measured by a matrigel invasion assay. Since extracellular signal‐regulated kinase (Erk) is involved in cellular movement and aberrant Akt activation contributes to increased cell invasiveness, we measured the effects of tick saliva on Akt and ERK activation by EGF. While EGF stimulation of ERK was not affected by tick saliva, Akt activation [j2]by 1 ng/ml EGF was inhibited ~55% (from 2.21+0.37 to 1.22+0.32). Our data indicate that a constituent(s) in tick saliva inhibits the invasiveness of an osteosarcoma cell and suggest that suppression of Akt signaling may be involved. Since metastasis of primary tumors often occurs in cancer, our data suggest the possibility of therapeutic agent(s) in tick saliva.

Clarithromycin inhibits fibroblast migration

The aim of the current study was to investigate the effect of 14-membered ring macrolide clarithromycin (CAM) on migration induced by human plasma fibronectin (HFn) or on contraction of human fetal lung fibroblasts (HFL-1). Using the blindwell chamber technique, CAM (10(-5) M) inhibited the migration of HFL-1 60.2+/-4.0% (p<0.05). Other antibiotics, such as ampicillin, minocycline or azithromycin had no effects on HFL-1 migration. The effect of CAM was concentration dependent. HFL-1 migration, stimulated by TXA(2) analog was also inhibited by CAM. Clarithromycin had no effect on HFL-1 mediated gel contraction that was another function of fibroblast at the wound area. Clarithromycin may contribute to the regulation of the wound healing response following injury by inhibiting fibroblast migration. These results could represent the therapeutic benefits of CAM.

Altered tissue repair in hevin‐null mice: Inhibition of fibroblast migration by a matricellular SPARC homolog

Matricellular proteins such as hevin, secreted protein acidic and rich in cysteine, and thrombospondin-2 play an important role during tissue repair through their influence on fundamental cellular activities such as adhesion, migration, proliferation, and extracellular matrix synthesis/reorganization. We have investigated the role played by hevin during excisional and incisional cutaneous wound repair in hevin-null mice. Hevin-null animals both close and heal their skin wounds faster than wild-type animals, as evidenced by enhanced macrophage infiltration of wound beds at early time points, the earlier appearance of mature extracellular matrix, and the overall higher maturity score. In addition, fibrovascular invasion of polyvinyl alcohol sponges was more robust in hevin-null mice, a result indicating that differences in cell migration might underlie the observed alterations in wound repair. Experiments in vitro showed that hevin induced the deadhesion and inhibited the migration of primary dermal fibroblasts in a Rac-1-dependent manner. These findings indicate that the differences in wound repair between hevin-null and wild-type animals can be attributed in part to the deadhesive function of hevin and reduced cell migration within dermal wound beds in which this protein is expressed.

Distinguishing fibroblast promigratory and procontractile growth factor environments in 3‐D collagen matrices

Understanding growth factor function during wound repair is necessary for the development of therapeutic interventions to improve healing outcomes. In the current study, we compare the effects of serum and purified growth factors on human fibroblast function in three different collagen matrix models: cell migration in nested matrices, floating matrix contraction, and stressed-released matrix contraction. The results of these studies indicate that platelet-derived growth factor (PDGF) is unique in its capacity to promote cell migration. Serum, lysophosphatidic acid, sphingosine-1-phophate (S1P), and endothelin-1 promote stressed-released matrix contraction but not cell migration. In addition, we found that S1P inhibits fibroblast migration and treatment of serum to remove lipid growth factors or treatment of cells to interfere with S1P(2) receptor function increases serum promigratory activity. Our findings suggest that different sets of growth factors generate promigratory and procontractile tissue environments for fibroblasts and that the balance between PDGF and S1P is a key determinant of fibroblast promigratory activity.

Regulation of fibroblast migration by tenascin-C

Synthesis of new tissue by fibroblasts is required for tissue rebuilding in response to injury. Fibroblast migration from surrounding healthy tissue into the fibrin-fibronectin provisional matrix deposited upon injury is a key rate-limiting step of this stage of tissue repair. These events must be tightly regulated. Excessive deposition of scar tissue is the major hallmark of fibrotic disease. Tenascin-C is an extracellular matrix glycoprotein that is transiently expressed upon tissue injury, where it is specifically localized to the wound edge, and persistently up-regulated in fibrotic disease. We have shown that full-length tenascin-C promotes fibroblast migration within fibrin-fibronectin matrices and we have mapped the domains within the molecule critical for enhancing migration. We also demonstrated that specific fragments of tenascin-C inhibit fibroblast migration. These results suggest that transient expression of tenascin-C at the wound boundary is key to tissue repair: its induction recruits fibroblasts into the wound and fragments resulting from its breakdown prevent excessive fibroblast infiltration. Our results demonstrate how fibroblast migration in three-dimensional provisional matrices may be differentially regulated by proteolysis of matrix molecules and could explain how persistent expression of tenascin-C contributes to the progression of fibrotic disease.

The Abl-related Gene Tyrosine Kinase Acts through p190RhoGAP to Inhibit Actomyosin Contractility and Regulate Focal Adhesion Dynamics upon Adhesion to Fibronectin

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.

Prostaglandin E2inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling

Uncontrolled fibroblast activation is one of the hallmarks of fibrotic lung disease. Prostaglandin E(2) (PGE(2)) has been shown to inhibit fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung. Understanding the mechanisms for these effects may provide insight into the pathogenesis of fibrotic lung disease. Previous work has focused on commercially available fibroblast cell lines derived from tissue whose precise origin and histopathology are often unknown. Here, we sought to define the mechanism of PGE(2) inhibition in patient-derived fibroblasts from peripheral lung verified to be histologically normal. Fibroblasts were grown from explants of resected lung, and proliferation and collagen I expression was determined following treatment with PGE(2) or modulators of its receptors and downstream signaling components. PGE(2) inhibited fibroblast proliferation by 33% and collagen I expression by 62%. PGE(2) resulted in a 15-fold increase in intracellular cAMP; other cAMP-elevating agents inhibited collagen I in a manner similar to PGE(2). These effects were reproduced by butaprost, a PGE(2) analog selective for the cAMP-coupled E prostanoid (EP) 2 receptor, but not by selective EP3 or EP4 agonists. Fibroblasts expressed both major cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1), but only a selective PKA agonist was able to appreciably inhibit collagen I expression. Treatment with okadaic acid, a phosphatase inhibitor, potentiated the effects of PGE(2). Our data indicate that PGE(2) inhibits fibroblast activation in primary lung fibroblasts via binding of EP2 receptor and production of cAMP; inhibition of collagen I proceeds via activation of PKA.

Prostaglandin E(2) inhibits fibroblast migration by E-prostanoid 2 receptor-mediated increase in PTEN activity.

An increased migratory phenotype exists in lung fibroblasts derived from patients with fibroproliferative lung disease. Prostaglandin E(2) (PGE(2)) suppresses fibroblast migration, but the receptor(s) and mechanism(s) mediating this action are unknown. Our data confirm that treatment of human lung fibroblasts with PGE(2) inhibits migration. Similar effects of butaprost, an E-prostanoid (EP) 2 receptor-specific ligand, implicate the EP2 receptor in migration-inhibitory signaling. Further, migration in fibroblasts deficient for the EP2 receptor cannot be inhibited by PGE(2) or butaprost, confirming the central role of EP2 in mediating these effects. Our previous data suggested that phosphatase and tensin homolog on chromosome ten (PTEN), a phosphatase that opposes the actions of phosphatidylinositol-3-kinase (PI3K), may be important in regulating lung fibroblast motility. We now report that both PGE(2) and butaprost increase PTEN phosphatase activity, without a concomitant increase in PTEN protein levels. This contributes to EP2-mediated migration inhibition, because migration in PTEN-null fibroblasts is similarly unaffected by EP2 receptor signaling. Increased PTEN activity in response to EP2 stimulation is associated with decreased tyrosine phosphorylation on PTEN, a mechanism known to regulate enzyme activity. Collectively, these data describe the novel mechanistic finding that PGE(2), via the EP2 receptor, decreases tyrosine phosphorylation on PTEN, resulting in increased PTEN enzyme activity and inhibition of fibroblast migration.

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration.

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

093 Prostaglandin E2 Inhibits Fibroblast Migration: Implications for Wound Healing

Background: Wound healing is a complex process involving multiple cell types, extracellular matrix components and soluble mediators. Prostaglandin E2 is an important component of the inflammatory response to injury. PGE2 can regulate the fibroblast response to injury via the EP receptor family. Here, we examine PGE2 regulation of fibroblast migration. Our analysis extends to fibroblasts representing a spectrum of wound healing phenotypes. Hypothesis: Prostaglandin E2 mediated inhibition of fibroblast migration is conserved across multiple fibroblast phenotypes. Methods: Primary cultures of human fetal, adult and keloid fibroblasts were used. Analysis of the EP receptor profile for each fibroblast phenotype was conducted using real‐time PCR, Western blot and immunohistochemistry. Fibroblast migration was quantified using a well established in vitro scratch assay. Results: Prostaglandin E2, via EP2/EP4 receptors, inhibits fibroblast migration in all fibroblast phenotypes. Fetal fibroblasts retain a more robust migratory phenotype when compared to normal adult and keloid fibroblasts. Normal adult fibroblasts exhibit a dramatic destabilization of the actin cytoskeleton which accompanies PGE2 inhibition of cell migration. This effect was not observed in fetal or keloid fibroblasts. Conclusions: Fibroblast activity in the wound bed can be altered by inflammatory mediators. The effects of prostaglandin E2 appear to be partially conserved across various fibroblast phenotypes. Variability in the response of these cells, however, indicates that fibroblasts derived from fetal tissue may retain intrinsic altered response mechanisms to endogenous inflammatory mediators.