Sphingosine 1-phosphate regulates peritoneal B-cell trafficking for subsequent intestinal IgA production

Sphingosine 1-phosphate (S1P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S1P. All peritoneal B cells including B1a, B1b, and B2 B cells express comparable levels of the type 1 S1P receptor. Thus, treatment with FTY720, an S1P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heat-killed Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S1P in connecting peritoneal B cells with intestinal B-cell immunity.     

Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson''s disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.     

Processive phosphorylation of ERK MAP kinase in mammalian cells

The mitogen-activated protein (MAP) kinase pathway is comprised of a three-tiered kinase cascade. The distributive kinetic mechanism of two-site MAP kinase phosphorylation inherently generates a nonlinear switch-like response. However, a linear graded response of MAP kinase has also been observed in mammalian cells, and its molecular mechanism remains unclear. To dissect these input-output behaviors, we quantitatively measured the kinetic parameters involved in the MEK (MAPK/ERK kinase)-ERK MAP kinase signaling module in HeLa cells. Using a numerical analysis based on experimentally determined parameters, we predicted in silico and validated in vivo that ERK is processively phosphorylated in HeLa cells. Finally, we identified molecular crowding as a critical factor that converts distributive phosphorylation into processive phosphorylation. We proposed the term quasi-processive phosphorylation to describe this mode of ERK phosphorylation that is operated under the physiological condition of molecular crowding. The generality of this phenomenon may provide a new paradigm for a diverse set of biochemical reactions including multiple posttranslational modifications.     

Epidermal growth factor system regulates mucin production in airways

Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor alpha (TGFalpha), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor alpha (TNFalpha). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNFalpha. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFalpha induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue-periodic acid-Schiff staining (reflecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNFalpha, EGF, or TGFalpha alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFalpha-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.     

1-磷酸鞘氨醇、鞘氨醇激酶与卒中

1-磷酸鞘氨醇(sphingosine 1-phosphate,S1P)是鞘磷脂的一种代谢产物,广泛存在于各种类型细胞.S1P既可作为细胞外介质,又能作为细胞内第二信使,参与细胞生长、存活、增殖、分化、Ca~(2+)动员等多种生理学过程.鞘氨醇激酶(sphingosine kinase,SPHK)是催化生成S1P的关键酶.S1P在中枢神经系统参与细胞凋亡、神经发生、免疫抑制、损伤修复等病理生理学过程,并与缺血性损伤、多发性硬化以及胶质细胞瘤等疾病密切相关.     

S-adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase

BACKGROUND & AIMS: After liver injury, hepatic S-adenosylmethionine (SAM) content decreases, and the blockage this molecule imposes on hepatocyte proliferation is released, facilitating liver regeneration. This activity of SAM is important for normal liver function because mice deficient in hepatic SAM display abnormal liver regeneration and develop hepatocellular carcinoma. How SAM regulates hepatocyte growth is unclear, but because SAM blocks hepatocyte growth factor (HGF)-induced cyclin D1 expression and DNA synthesis without affecting HGF-induced extracellular signal-regulated kinase phosphorylation, the mitogen-activated protein kinase (MAPK) pathway is probably not the target. METHODS: The effects of SAM on AMPK, HuR localization were assessed in rat hepatocytes after HGF, AICAR, and SAM treatment. RESULTS: We show here that HGF and 5-aminoimidazole-4-carboxamide-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), induce the phosphorylation of AMPK in hepatocytes and that SAM blocks this process. We also show that HGF- and AICAR-induced AMPK activation stimulate the transport from nucleus to cytoplasm of HuR, an RNA-binding protein that increases the half-life of target mRNA such as cyclin A2, and that SAM blocks this process. We found that, in hepatocytes, AICAR increases HuR binding to cyclin A2 messenger RNA (mRNA) as well as the expression and stability of this mRNA and that SAM blocks these events. Consistently, we found that AICAR induces hepatocyte proliferation and that SAM blocks this effect. Finally, we found that liver AMPK phosphorylation, cytoplasmic HuR, and binding of HuR to HuR-target mRNA and the steady-state levels of these mRNA are increased in knockout mice deficient in hepatic SAM. CONCLUSIONS: Our results yield novel insights about the mechanism by which SAM inhibits cell-cycle progression in the liver.     

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways

Purpose

Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified.

Methods

SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA.

Results

Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580.

Conclusions

SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.     

Sphingosine kinase 1 participates in insulin signalling and regulates glucose metabolism and homeostasis in KK/Ay diabetic mice

Aims/hypothesis The aim of this study was to determine the potential role of sphingosine kinase 1 (SPHK1), a key sphingolipid metabolic enzyme, in glucose metabolism and homeostasis. Methods SMMC-7721 hepatoma cells and C2C12 myotube cells were used to explore the role of SPHK1 in glucose uptake in vitro. KK/Ay type 2 diabetic mice, which were transfected with adenovirus harbouring the human SPHK1 gene by i.v. injection, were used to investigate the glucose-lowering effects of SPHK1 in vivo. Results The basal glucose uptake and the insulin-stimulated glucose uptake in both 7721 cells and C2C12 cells were markedly enhanced when SPHK1 was overexpressed by adenovirus-mediated gene transfer, whereas they were substantially reduced when the expression of SPHK1 was inhibited or the activity of SPHK1 was blocked. Insulin could activate SPHK1 of both cell lines in a dose-dependent manner. SPHK1 gene delivery significantly reduced the blood glucose level of KK/Ay diabetic mice, but had no effect on that of normal animals. It also attenuated elevated levels of plasma insulin, NEFA, triacylglycerol, cholesterol and LDL, significantly ameliorated hyperglycaemia-induced injury of liver, heart and kidney, and enhanced phosphorylation of insulin-signalling kinases such as Akt and glycogen synthase kinase 3β in livers of the diabetic animals. Conclusions/interpretation SPHK1 is involved in insulin signalling and plays an important role in the regulation of glucose and fat metabolism; adenovirus-mediated SPHK1 gene transfer might provide a novel strategy in the treatment of type 2 diabetes mellitus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. M. M. Ma and J. L. Chen have contributed equally to this work.     

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca^2 +-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca^2 +-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca^2 +. These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β.     

Protein kinase Cdelta-dependent phosphorylation of syndecan-4 regulates cell migration

Endothelial cell (EC) migration is a complex process requiring exquisitely coordinated focal adhesion assembly and disassembly. Protein kinase C (PKC) is known to regulate focal adhesion formation. Because lysophosphatidylcholine (lysoPC), a major lipid constituent of oxidized low-density lipoprotein, can activate PKC and inhibit EC migration, we explored the signaling cascade responsible for this inhibition. LysoPC increased PKCdelta activity, measured by in vitro kinase activity assay, and increased PKCdelta phosphorylation. Decreasing PKCdelta activation, using pharmacological inhibitors or antisense oligonucleotides, diminished the antimigratory effect of lysoPC. LysoPC-induced PKCdelta activation was followed by increased phosphorylation of the transmembrane proteoglycan, syndecan-4, and decreased binding of PKCalpha to syndecan-4, with a concomitant decrease in PKCalpha activity. A reciprocal relationship was noted between the interaction of PKCalpha and alpha-actinin with syndecan-4. These changes were temporally related to the observed changes in cell morphology and the inhibition of migration of ECs incubated with lysoPC. The data suggested that generalized activation of PKCdelta by lysoPC initiated a cascade of events, including phosphorylation of syndecan-4, displacement and decreased activity of PKCalpha, binding of alpha-actinin to syndecan-4, and disruption of the time- and site-specific regulation of focal adhesion complex assembly and disassembly required for normal cell migration.     

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.     

Sphingosine 1-phosphate receptor 2 negatively regulates neointimal formation in mouse arteries

Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P2)-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P2-null but not in wild-type arteries. This was accompanied with a significant increase in both medial and intimal smooth muscle cell (SMC) replication between days 4 to 28, with only minimal replication in wild-type arteries. S1P2-null SMCs showed a significant increase in migration when stimulated with S1P alone and together with platelet-derived growth factor, whereas both wild-type and null SMCs migrated equally well to platelet-derived growth factor. S1P increased Rho activation in wild-type but not in S1P2-null SMCs, and inhibition of Rho activity promoted S1P-induced SMC migration. Plasma S1P levels were similar and did not change after surgery. These results suggest that activation of S1P2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development.     

PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF,P-Rex1

The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.The phosphoinositide 3-kinase (PI3K) family of lipid kinases plays a prominent role in the growth and survival of several types of cancer (1). The PI3K pathway is aberrantly activated by a number of different mechanisms in cancers. These include genetic mutation and/or amplification of key pathway components, such as amplification or mutation of the PI3K catalytic subunit p110α (encoded by PIK3CA gene), mutation or deletion of the phosphatase PTEN, amplification or mutation of the gene encoding for the PI3K effector protein kinase AKT, as well as constitutive activation of receptor tyrosine kinases (RTKs) (e.g., HER2 amplification in breast cancer) or other less frequent events (2). PI3K phosphorylates the phosphoinositide PI(4,5)P2 in the 3′OH group of the inositol ring to produce PI(3,4,5)P3. PI(3,4,5)P3 directly binds to the pleckstrin homology (PH) domains of certain proteins, such as AKT, leading to their activation, which in turn transmit growth and survival signals.These findings have encouraged the development of several different PI3K inhibitors, many of which are either in or approaching clinical trial testing. Genotype-driven patient selection has been investigated to uncover patient populations that will be particularly susceptible to PI3K inhibitors. Cancers harboring mutations in the PIK3CA gene have emerged as among the most sensitive to single-agent PI3K inhibitors in several preclinical studies, although clinical activity to date has been mixed (3–6). These gain-of-function mutations in the PI3KCA gene are found in a broad range of cancers, and they are highly enriched in breast cancer, where they are observed in 20–25% of cases (7). In addition, breast cancers with amplified HER2, which comprise ∼20% of all breast cancers, (8) are also particularly sensitive to PI3K inhibition (9–11). However, even among patients whose cancers harbor PIK3CA mutations, a significant heterogeneity of responses has been observed to PI3K inhibitors currently being tested in clinical studies (3–5). There have been some patients with bona fide response evaluation criteria in solid tumors (RECIST) criteria responses, but the majority has not had similarly impressive outcomes. These early clinical results highlight the potential utility of a biomarker of sensitivity to single-agent PI3K inhibitors.Interestingly, early clinical trial reports have found that inhibition of PI3K signaling may sometimes lead to suppression of protein kinase MEK (MEK)/ERK signaling (6). Although a previous laboratory study had shown that the PI3K/mammalian target of rapamycin (mTOR) inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin can inhibit protein kinase RAF (RAF)/MEK/ERK-signaling (12), this clinical observation was initially surprising because several studies have shown that inhibitors of components of the PI3K signaling pathway (such as AKT and mTOR inhibitors) actually lead to activation of the MEK/ERK signaling in many cancer types (11, 13), and such feedback activation may impair sensitivity to PI3K pathway inhibitors (9, 11, 14). Because PIK3CA and HER2 amplified breast cancers are particularly sensitive to single-agent PI3K pathway inhibitors, we investigated how PI3K inhibitors impact MEK/ERK signaling in these genetically defined subsets of breast cancers. In our study, we found that several cell lines harboring PIK3CA mutation and/or HER2 amplification suppress MEK/ERK pathway signaling as well as the AKT pathway after treatment with PI3K inhibitors, and importantly, inhibition of both pathways is necessary for maximal antitumoral activity. Moreover we identify that the mechanistic link between PI3K and MEK/ERK is via a PI(3,4,5)P3-dependent regulation of the phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1)/ small GTPase Rac1 (Rac1)/protein kinase c-RAF (c-RAF) pathway in these cancers. Importantly, the expression levels of the Rac guanine exchange factor (Rac-GEF), P-Rex1, correlate with sensitivity to PI3K inhibitors in these breast cancer cell lines.     

The synthetic metalloproteinase inhibitor batimastat suppresses injury-induced phosphorylation of MAP kinase ERK1/ERK2 and phenotypic modification of arterial smooth muscle cells in vitro

Smooth muscle cell (SMC) migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. The extracellular matrix has important functions in modulating SMC structure and function, but less is known about the role of the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors. The present study investigates the effects of the synthetic MMP inhibitor batimastat (BB94) on vascular SMCs. As experimental model, rat aortic smooth muscle cells in primary and secondary cultures were employed. Electron microscopy was used to investigate the effects of BB94 on the overall phenotypic properties of the cells. Induction of DNA synthesis and migration was studied by thymidine autoradiography and counting of cells moving into an injured zone. Gelatin zymography was used for the detection of BB94-mediated inhibition of injury-induced MMP activity. Phosphorylation of the mitogen-activated protein kinases ERK1/ERK2, two potential mediators of the injury-induced activation of the cells, was measured by Western blotting. The results show that BB94 restrained the phenotypic modulation of vascular SMCs in primary cultures and suppressed injury-induced DNA synthesis and migration. Moreover, the upregulation of ERK1/ERK2 phosphorylation in injured secondary cultures and in cells treated with bFGF was markedly reduced by BB94, whereas TIMP-2 lacked a clear effect. Our data suggest that BB94 inhibits injury-induced activation of vascular SMCs by acting on MMPs as well as other targets.