Non-steroidal anti-inflammatory drugs suppress the ERK signaling pathway via block of Ras/c-Raf interaction and activation of MAP kinase phosphatases

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit cancer cell growth, induce apoptosis and decrease tumor metastasis. We have previously reported that a NSAID NS398 repressed the expression of matrix metalloproteinase-2 (MMP-2) via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we investigate the underlying mechanism of this inhibition. In vitro kinase assay indicated that NS398 could not directly inhibit c-Raf, MEK1 and ERK enzymatic activity. We found that NS398 increased the inhibitory phosphorylation of Ser259 in c-Raf, attenuated membrane recruitment of c-Raf and inhibited Ras/c-Raf interaction to attenuate activation of this kinase. This is a general effect for NSAIDs because sulindac sulfide, aspirin and indomethacin also inhibited the binding of c-Raf to Ras. Immunofluorescent staining verified that NS398 reduced the serum-induced membrane recruitment of c-Raf in cells. However, overexpression of constitutively active c-Raf only partly reversed NS398-induced inhibition of MMP-2 expression. Interestingly, we found that NS398 up-regulated the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and MKP-3. Block of MKP activity by sodium orthovanadate also partly counteracted the inhibitory effect of NS398. Overexpression of constitutively active c-Raf and treatment of sodium orthovanadate together completely reversed the inhibition of MMP-2 by NS398. Taken together, we conclude that NS398 and other NSAIDs act via inhibition of Ras/c-Raf interaction and up-regulation of MKPs to suppress the ERK-mediated signaling.     

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.     

NS398抑制宫颈癌Hela细胞增殖及MMP2的表达

目的：NS398是非甾体类抗炎药物的一种,它可以抑制肿瘤细胞增殖,诱导肿瘤细胞凋亡,但其在肿瘤发展中的作用机制尚不清楚。宫颈癌是女性最常见的恶性肿瘤,其侵袭转移是患者死亡的主要原因。基质金属蛋白酶几乎能降解细胞外基质中的各种蛋白质成分,在肿瘤侵袭转移中起着十分关键的作用。本文则以宫颈癌Hela细胞为研究对象,观察NS398对Hela细胞的增殖及其基质金属蛋白酶2表达的影响,探讨NS398在宫颈癌侵袭转移过程中的作用及可能机制。方法：用不同浓度的NS398（O、25、50、75、100μmol／L）处理宫颈癌Hela细胞48小时,以噻唑蓝（MTT）比色法分析细胞生长抑制率,酶联免疫吸附实验（EuSA）与蛋白免疫印迹技术分别检测MMP2活性及蛋白表达的变化。结果：不同浓度的NS398处理Hela细胞后,MTT法分析显示,NS398可明显降低细胞代谢MTT的能力（P〈0．05）;ELISA检测发现,NS398可减少细胞培养液中活性MMP2含量（P〈0．05）;Western免疫印迹检测显示,NS398可下调细胞MMP2蛋白的表达（P〈0．05）,这些效应均呈剂量依赖性。结论：Ns398呈剂量依赖性抑制Hela细胞的增殖,抑制细胞中MMP2的活性,并下调细胞MMP2的表达。结果提示,NS398可通过抑制肿瘤细胞的增殖而抑制宫颈癌的生长,通过抑制MMP2的活性和下调MMP2蛋白的表达而抑制宫颈癌的侵袭转移,这为NS398在宫颈癌防治中的应用提供了新的实验依据。     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.

Limited clinical and experimental studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) may inhibit gastric cancer growth. However, the mechanisms involved are not completely understood and cannot be explained by COX-2 inhibition alone. MAPK signaling pathway is essential for cell proliferation, but the effect of NSAIDs on MAPK activity and phosphorylation in gastric cancer has never been studied. Since increased and unregulated cell proliferation and reduced cell apoptosis are important features of cancer growth, we studied whether NS-398, a selective COX-2 inhibitor and/ or indomethacin (IND), a non-selective NSAID: 1) inhibit gastric cancer cell proliferation, 2) whether this inhibition is mediated via MAPK (ERK2), and 3) whether NSAIDs enhance apoptosis in gastric cancer cells. Human gastric epithelial cells (MKN28) derived from gastric tubular adenocarcinoma were cultured and treated with either vehicle, IND (0.25-0.5mM) or NS-398 (50-100 microM) for 6, 16, 24 and 48h. Studies: 1) Cellular proliferation was determined by 3H-thymidine uptake. 2) MAPK activity was measured by incorporation of radiolabeled phosphate into myelin basic protein. 3) Apoptosis was evaluated using TUNEL assay. IND and NS-398 significantly inhibited the proliferation of MKN28 cells at 24h by 3.5 - 5 fold (p<0.002) and at 48h by 2.5 - 10 fold (p<0.02). Both NSAIDs also significantly inhibited ERK2 activity: IND >53% inhibition, NS-398, 100 microM >72% inhibition; all p<0.05. Both IND and NS-398 significantly increased apoptotic index. In conclusion, IND and NS-398 significantly inhibit proliferation and growth of human gastric cancer cell line MKN28. This effect is mediated by NSAID-induced inhibition of MAPK (ERK2) kinase signaling pathway, essential for cell proliferation. NSAIDs also increase apoptosis in MKN28 cells. In addition to inhibiting cyclooxygenase, NSAIDs inhibit phosphorylating enzymes--kinases essential for signaling cell proliferation.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

Restoration of thrombospondin 1 expression in tumor cells harbouring mutant ras oncogene by treatment with low doses of doxycycline

Oncogenes act as inducers of tumor neovascularization, at least in part through suppression of endogenous angiogenesis inhibitors, e.g., thrombospondin 1 (TSP-1/THSP1). Therefore, restoration of TSP-1 levels can be viewed as a possible means to inhibit tumor angiogenesis. We observed that low concentrations (0.1-10 microg/ml) of doxycycline (but not those of related tetracycline) restore TSP-1 expression in H-ras oncogene-expressing tumor cell lines (528ras1, MT-Ras). Interestingly, this effect was relatively ras-specific, as doxycycline did not alter TSP-1 expression in several cell lines (e.g., 528neu2 fibrosarcoma, B16F1 melanoma, and Lewis lung carcinoma) harbouring other types of transforming alterations. Doxycycline-induced reversal of TSP down-regulation was abrogated under hypoxic conditions. Therefore, we conclude that, in vivo, TSP-1 is likely under dual and/or synergistic control of oncogenes and hypoxia-related pathways. Disruption of both components may be necessary for the 'rescue' of TSP-1 expression in ras-driven cancers.     

Age-related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase-2 and PPAR gamma expression and is altered by long-term NS398 administration

Age-related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age-related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long-term (12-month) oral administration of nicotine and/or the cyclooxygenase-2 (COX-2) preferring non-steroidal anti-inflammatory drug (NSAID) NS398 on nAChR alpha4, alpha5, alpha7, and beta4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX-2 and the peroxisome proliferator-antagonist receptor subtype gamma-2 (PPAR gamma2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChR alpha4 and to a lesser extent nAChR beta4, but not nAChR alpha5 or alpha7, but nicotine exhibited no similar effect. Nicotine and NS398 co-administration abolished the NS398-related effect on nAChR alpha4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined.     

Protective roles of the fractalkine/CX3CL1-CX3CR1 interactions in alkali-induced corneal neovascularization through enhanced antiangiogenic factor expression

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.     

Age‐related loss of neuronal nicotinic receptor expression in the aging mouse hippocampus corresponds with cyclooxygenase‐2 and PPARγ expression and is altered by long‐term NS398 administration

Age‐related changes in the mammalian dorsal hippocampus are associated with diminished expression of neuronal nicotinic acetylcholine receptors (nAChR), which is particularly severe in pathologies such as those associated with dementias, including Alzheimer's disease. Because the mouse is a useful model for age‐related decline in nAChR expression in the basal forebrain and limbic system, we used immunohistochemistry to examine the influence of long‐term (12‐month) oral administration of nicotine and/or the cyclooxygenase‐2 (COX‐2) preferring non‐steroidal anti‐inflammatory drug (NSAID) NS398 on nAChRα4, α5, α7, and β4 expression in the C57BL/6 mouse. Inhibitory neurons of the dorsal hippocampus that express nAChRs also constitutively express COX‐2 and the peroxisome proliferator‐antagonist receptor subtype gamma‐2 (PPARγ2) which is also a target of NS398. Administration of NS398 correlated with retention of nAChRα4 and to a lesser extent nAChRβ4, but not nAChRα5 or α7, but nicotine exhibited no similar effect. Nicotine and NS398 co‐administration abolished the NS398‐related effect on nAChRα4 retention. These results provide evidence that the interaction during aging between oral administration of nicotine and NSAIDs are not straightforward and could even be antagonistic when combined. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Interactions between CD47 and thrombospondin reduce inflammation

CD47 on the surface of T cells was shown in vitro to mediate either T cell activation or, in the presence of high amounts of thrombospondin (TSP), T cell apoptosis. We report here that CD47-deficient mice, as well as TSP-1 or TSP-2-deficient mice, sustain oxazolone-induced inflammation for more than four days, whereas wild-type mice reduce the inflammation within 48 h. We observe that prolonged inflammation in CD47-, TSP-1-, or TSP-2-deficient mice is accompanied by a local deficiency of T cell apoptosis. Finally, we show that upon activation normal T cells increase the expression of the proapoptotic Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein) and undergo CD47-mediated apoptosis. This finding is consistent with our previous demonstration of a physical interaction between BNIP3 and CD47 that inhibits BNIP3 degradation by the proteasome, sensitizing T cells to CD47-induced apoptosis. Overall, these results reveal an important role in vivo for this new CD47/BNIP3 pathway in limiting inflammation by controlling the number of activated T cells.     

Downregulation of SPARC Expression Inhibits the Invasion of Human Trophoblast Cells In Vitro

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

Cyclooxygenase inhibitors not inhibit resting lung cancer A549 cell proliferation

Cyclooxygenase (COX) inhibitors were regarded as anticarcinogenic agents for lung cancer at least partly via PGE2; but these were based on cytokin stimulation experiment on A549 cell. In order to clarify whether COX inhibitors directly inhibit A549 cell, three COX inhibitors, NS398 (selective COX-2 inhibitor), SC560 (selective COX-1 inhibitor), and acetyl salicylic acid (ASA, non-selective COX inhibitor), were studied. NS398, and ASA, can inhibit PGE2 generation via COX-2 inhibition. The viability of A549 cell was assayed by MTT. However, without cytokin stimulation, all the three inhibitors (NS398 0.2-20 microM; SC560 1.0-100 nM; ASA 0.01-1.0 mM) were not able to inhibit A549 cell proliferation, in the other way round, NS398 promoted cell growth. And arachidonic acid (AA) and lipopolysaccharide (LPS) did not disturb the property of its growth. These data suggested that without cytokin stimulation, COX and PGE2 may not be the kernel molecules involved in A549 cell proliferation, and COX inhibitors could not inhibit A549 cell growth directly.     

Induction of Egr-1 is associated with anti-metastatic and anti-invasive ability of beta-lapachone in human hepatocarcinoma cells

beta-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of beta-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. beta-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that beta-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in beta-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that beta-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.     

Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.     

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.