Association of aorta intima permeability with myosin light chain kinase expression in hypercholesterolemic rabbits

The development of hypercholesterolemia is a multifactorial process in which elevated plasma cholesterol levels play a central role. This study analyzed the variability of the expression and activity of myosin light chain kinase (MLCK) and endothelial permeability in the artery wall of rabbits after feeding the animals with a normal or a high-cholesterol diet. Hypercholesterolemia was induced by a high-cholesterol diet for 4 weeks. Aortas were removed and analyzed for endothelial permeability and MLCK expression. Samples of the arterial media were analyzed for MLCK activity and expression. A selective MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in hypercholesterolemia rabbit (1 mg/kg body weight). The aortas of high-cholesterol diet rabbits showed an increase in MLCK expression and activity (nearly threefold compare with control) as well as endothelial permeability. ML7 inhibit MLC phosphorylation and MLCK activity (nearly twofold compare with control) and endothelial permeability stimulated by cholesterol. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and activity through which endothelial permeability is increased.     

MicroRNA-147b Regulates Vascular Endothelial Barrier Function by Targeting ADAM15 Expression

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3′ UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.     

Myosin light chain kinase mediates intestinal barrier disruption following burn injury

Background

Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.

Methodology/Principal Findings

Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.

Conclusions/Significance

The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.     

MicroRNA-492 reverses high glucose-induced insulin resistance in HUVEC cells through targeting resistin

The development of atherosclerosis (AS) is a multifactorial process, in which elevated plasma resistin (a key factor leading to insulin resistance) levels play an important role. Emerging evidence indicate that microRNAs (miRNAs) are involved in AS; However, the regulation and function of miRNAs in response to AS remain poorly understood. Our study analyzed the effects of miR-492 on insulin resistance, endothelial activation, and resistin expression in apoE knock-out mice and human umbilical vein endothelial cells after high-glucose treatment and miR-492 mimics transfection. We also investigated the underlying molecular mechanisms. Our results showed that high glucose stress induced a significant decrease in miR-492 expression, with a remarkable upregulation of resistin expression. We then identified resistin as a novel direct target of miR-492 using 3′-UTR luciferase reporter assay. Histopathologic examination demonstrated that upregulation of miR-492 attenuated endothelial cells migration and lipid accumulation induced by high glucose stress. Further investigation demonstrated that the upregulation of p-STAT3, SOCS, and P-selectin activation induced by high glucose stress was attenuated by upregulation of miR-492. Together, our findings indicate that miR-492 contributes to insulin resistance and endothelial dysfunction induced by high glucose, via directly downregulating resistin expression, and involving STAT3 phosphorylation, SOCS, and P-selectin activation.     

EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption

Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia. Inflammation is initiated by activation of nuclear factor (NF)-kappaB, and paracellular permeability is enhanced via a Ca2+- and myosin light-chain kinase (MLCK)-dependent pathway. The aims of this study were to identify signaling pathways by which EPEC triggers inflammation and to determine whether these pathways parallel or diverge from those that alter permeability. EPEC-induced phosphorylation and degradation of the primary inhibitor of NF-kappaB (IkappaBalpha) were tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta independent. In contrast to Salmonella typhimurium, EPEC-stimulated IkappaBalpha degradation and IL-8 expression did not require Ca2+. Instead, extracellular signal-regulated kinase (ERK)-1/2 was significantly and rapidly activated. ERK1/2 inhibitors attenuated IkappaBalpha degradation and IL-8 expression. Although ERK1/2 can activate MLCK, its inhibition had no impact on EPEC disruption of the tight junction barrier. In conclusion, EPEC-induced inflammation 1) is TNF-alpha and IL-1beta receptor independent, 2) utilizes pathways differently from S. typhimurium, 3) requires ERK1/2, and 4) employs signals that are distinct from those that alter permeability. This is the first time that EPEC-activated signaling cascades have been linked to independent functional consequences.     

Deletion of myosin light chain kinase in endothelial cells has a minor effect on the lipopolysaccharide-induced increase in microvascular endothelium permeability in mice

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)-mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS-induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild-type mice. However, the LPS-induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS-mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.     

Induction of SPARC by VEGF in human vascular endothelial cells

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.     

p38 MAP kinase-dependent regulation of endothelial cell permeability

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.     

MicroRNA‐21 regulates intestinal epithelial tight junction permeability

Increased tight junction (TJ) barrier permeability, induced by tumour necrosis factor (TNF)‐α, may lead to the defects in TJ barrier and subsequent development of inflammation. Recent evidence suggests that miR‐21 is implicated in inflammatory diseases. However, the physiological role of miR‐21 in intestinal permeability remains elusive. This study aimed to explore the role of miR‐21 in intestinal epithelial tight junction permeability. The filter‐grown Caco‐2 monolayers model system was established to mimic intestinal barrier defect. The tight junction proteins were detected by immunofluorescence and western blot analysis. The expression of miR‐21 was assessed by real‐time polymerase chain reaction (PCR). We found that the expression of miR‐21 was increased significantly in TNF‐α induced intestinal TJ barrier defect model. miR‐21 overexpression significantly enhanced while miR‐21 knockdown significantly decreased intestinal permeability. In addition, miR‐21 overexpression significantly increased while miR‐21 knockdown significantly decreased the levels of interleukin‐6, interleukin‐8 and prostaglandin E2 in cell culture medium. Furthermore, miR‐21 positively regulated Akt phosphorylation and negatively regulated Phosphatase and tensin homolog (PTEN) expression in Caco‐2 cells. Our results suggest that miR‐21 may regulate intestinal epithelial tight junction permeability through PTEN/PI3K/Akt signalling pathway. This promotes the feasibility of targeting miR‐21 in the clinical to preserve the intestinal barrier. Copyright © 2015 John Wiley & Sons, Ltd.     

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

Store-operated calcium entry and increased endothelial cell permeability

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.     

MicroRNA-93 Downregulation Ameliorates Cerebral Ischemic Injury Through the Nrf2/HO-1 Defense Pathway

The present study was designed to evaluate the potential role of miR-93 in cerebral ischemic/reperfusion (I/R) injury in mice. The stroke model was produced in C57BL/6 J mice via middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. And miR-93 antagomir was transfected to down-regulate the miR-93 level. Our results showed that miR-93 levels in the cerebral cortex of mice increased at 24 and 48 h after reperfusion. Importantly, in vivo study demonstrated that treatment with miR-93 antagomir reduced cerebral infarction volume, neural apoptosis and restored the neurological scores. In vitro study demonstrated that miR-93 antagomir attenuated hydrogen peroxide (H₂O₂)-induced injury. Moreover, miR-93 antagomir suppressed oxidative stress in I/R brain and H₂O₂ treated cortical neurons. Furthermore, we founded that down-regulation of miR-93 increased the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) and the luciferase reporter assay confirmed that miR-93 directly binds to the predicted 3′-UTR target sites of the nrf2 gene. Finally, we found that knockdown of Nrf2 or HO-1 abolished miR-93 antagomir-induced neuroprotection against oxidative stress in H₂O₂ treated neuronal cultures. These results suggested that miR-93 antagomir alleviates ischemic injury through the Nrf2/HO-1 antioxidant pathway.     

A chronic high-cholesterol diet paradoxically suppresses hepatic CYP7A1 expression in FVB/NJ mice

Cholesterol 7α-hydroxylase (CYP7A1) encodes for the rate-limiting step in the conversion of cholesterol to bile acids in the liver. In response to acute cholesterol feeding, mice upregulate CYP7A1 via stimulation of the liver X receptor (LXR) α. However, the effect of a chronic high-cholesterol diet on hepatic CYP7A1 expression in mice is unknown. We demonstrate that chronic cholesterol feeding (0.2% or 1.25% w/w cholesterol for 12 weeks) in FVB/NJ mice results in a >60% suppression of hepatic CYP7A1 expression associated with a >2-fold increase in hepatic cholesterol content. In contrast, acute cholesterol feeding induces a >3-fold upregulation of hepatic CYP7A1 expression. We show that chronic, but not acute, cholesterol feeding increases the expression of hepatic inflammatory cytokines, tumor necrosis factor (TNF)α, and interleukin (IL)-1β, which are known to suppress hepatic CYP7A1 expression. Chronic cholesterol feeding also results in activation of the mitogen activated protein (MAP) kinases, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, we demonstrate in vitro that suppression of CYP7A1 by TNFα and IL-1β is dependent on JNK and ERK signaling. We conclude that chronic high-cholesterol feeding suppresses CYP7A1 expression in mice. We propose that chronic cholesterol feeding induces inflammatory cytokine activation and liver damage, which leads to suppression of CYP7A1 via activation of JNK and ERK signaling pathways.     

IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.     

Endothelial microparticles-mediated transfer of microRNA-19b promotes atherosclerosis via activating perivascular adipose tissue inflammation in apoE−/− mice

Microparticles(MPs) are the major carriers of circulating microRNAs. Our previous study has shown that microRNA (miR)-19b in endothelial cell-derived microparticles (EMPs) is significantly increased in patients with unstable angina. However, little is known about the relationship between miR-19b in EMPs and the progression of atherosclerosis. The aim of the present study was to define the role and potential mechanism of miR-19b incorporated in EMPs in the development of atherosclerosis.Western-diet-fed apoE^?/? mice were injected with phosphate buffered solution(PBS), EMP carrying microRNA control(EMP^control) or miR-19b mimic (EMP^miR19b) intravenously. Systemic treatment with EMP^miR19b significantly accelerated carotid artery atherosclerosis progression by increasing lipid, macrophages and smooth muscle cells and decreasing collagen content in atherosclerotic plaque. Fluorescence-labelled EMP^miR19b injection proved that miR-19b could be transported into perivascular adipose tissue(PVAT) by EMPs. EMP^miR19b treatment also promoted inflammatory cytokines secretion and macrophages infiltration in PVAT. In further experiment, apoE^?/? mice were divided into 3 groups: EMP^controlPVAT(+), EMP^miR19bPVAT(+) and EMP^miR19bPVAT(-), based on removing or keeping pericarotid adipose tissue and injected with EMP^control or EMP^miR19b. Loss of PVAT attenuated EMP^miR19b-mediated effects on increasing carotid atherosclerosis formation and inflammatory cytokines level in plaque. EMP^miR19b inhibited suppressor of cytokine signaling 3 (SOCS3) expression in PVAT. Our findings demonstrate that miR-19b in EMPs exaggerates atherosclerosis progression by augmenting PVAT-specific inflammation proceeded by downregulating SOCS3 expression.     

Simvastatin Ameliorates Matrix Stiffness-Mediated Endothelial Monolayer Disruption

Arterial stiffening accompanies both aging and atherosclerosis, and age-related stiffening of the arterial intima increases RhoA activity and cell contractility contributing to increased endothelium permeability. Notably, statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors whose pleiotropic effects include disrupting small GTPase activity; therefore, we hypothesized the statin simvastatin could be used to attenuate RhoA activity and inhibit the deleterious effects of increased age-related matrix stiffness on endothelial barrier function. Using polyacrylamide gels with stiffnesses of 2.5, 5, and 10 kPa to mimic the physiological stiffness of young and aged arteries, endothelial cells were grown to confluence and treated with simvastatin. Our data indicate that RhoA and phosphorylated myosin light chain activity increase with matrix stiffness but are attenuated when treated with the statin. Increases in cell contractility, cell-cell junction size, and indirect measurements of intercellular tension that increase with matrix stiffness, and are correlated with matrix stiffness-dependent increases in monolayer permeability, also decrease with statin treatment. Furthermore, we report that simvastatin increases activated Rac1 levels that contribute to endothelial barrier enhancing cytoskeletal reorganization. Simvastatin, which is prescribed clinically due to its ability to lower cholesterol, alters the endothelial cell response to increased matrix stiffness to restore endothelial monolayer barrier function, and therefore, presents a possible therapeutic intervention to prevent atherogenesis initiated by age-related arterial stiffening.     

Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts

Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.