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Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells
Matrix Biology 26 (2007) 463 – 472 www.elsevier.com/locate/matbio
Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells Min Wu a,1 , Hamid Massaeli b,2 , Melanie Durston a , Nasrin Mesaeli a,⁎ a
Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, Department of Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada b Institute of Clinical Research of Montréal, Montreal, Canada Received 28 February 2006; received in revised form 6 February 2007; accepted 23 February 2007
Abstract Calreticulin is an endoplasmic reticulum protein important in cardiovascular development. Deletion of the calreticulin gene leads to defects in the heart and the formation of omphaloceal. These defects could both be due to changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of the MMPs which are essential for cardiovascular remodelling and development. Here, we tested the hypothesis that the defects observed in the heart and body wall of the calreticulin null embryos are due to alterations in MMP-2 and MMP-9 activity. Our results demonstrate that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells. We also showed that there is a significant increase in the expression level of membrane type-1 matrix metalloproteinase (MT1-MMP). In contrast, there was no change in the tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2 in the calreticulin deficient cells as compared to the wild type cells. Interestingly, the inhibition of the MEK kinase pathway using PD98059 attenuated the decrease in the MMP-9 mRNA with no effect on the MMP-2 mRNA level in the calreticulin deficient cells. Furthermore, PI3 kinase inhibitor decreased the expression of both the MMP-2 and MMP-9. This study is the first report on the role of calreticulin in regulating MMP activity. © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Calreticulin; Matrix metalloproteinase-2; MMP-9; MT1-MMP; PI3 kinase; MEK
1. Introduction Extracellular matrix (ECM) is an important structure within tissues that provides a supportive environment for cellular growth, development and morphogenesis. Changes in the ECM
Abbreviations: MMP, matrix metalloproteinase; CRT, calreticulin, crt−/−, calreticulin deficient; wt, wild type; MT1-MMP, membrane type-1 matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; ECM, extracellular matrix; PI3 kinase, phosphatidylinositol 3 kinase. ⁎ Corresponding author. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Tel.: +1 204 235 3671; fax: +1 204 231 1151. E-mail address: [email protected] (N. Mesaeli). 1 Current address: The cardiovascular department of the first affiliated hospital of Medical College of Shantou University, Shantou, Guangdong, 515041, the People's Republic of China. 2 Current address: Institute of Cardiovascular Sciences, St. Boniface Research Centre, Winnipeg, Manitoba, Canada.
composition are important steps associated with the processes of embryogenesis, tissue remodelling, cancer cell invasion, metastasis and angiogenesis (Egeblad and Werb, 2002; McCawley and Matrisian, 2000). Matrix metalloproteinases (MMPs) are a superfamily of zinc-binding endopeptidases that degrade the ECM. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are two members of MMPs which degrade native collagen type IV, collagen type V, fibronectin, entactin, and elastin (Nagase and Woessner, 1999). They play an important role in many processes which lead to cardiovascular disease such as vascular remodelling (Li et al., 2000), atherosclerotic plaque instability, heart failure (Spinale et al., 2000), development of aneurysms (Pyo et al., 2000), myocardial infarction, and left ventricular remodelling after myocardial infarction (Creemers et al., 2001). MMP-2 and MMP-9 are synthesized as pro-enzymes which are activated upon proteolysis by different mechanisms including both plasmin-independent and plasmin-dependent
0945-053X/$ - see front matter © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2007.02.005
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Fig. 1. Analysis of MMP-2 and MMP-9 activity in wild type (wt) and CRT deficient (crt−/−) mouse embryonic fibroblast cells (MEF) using gelatin zymography. Cells were cultured in conditioned media for 48 h, then the media were collected and zymography was carried out (as described in Experimental procedures). (A) Upper panel shows a representative zymography of the conditioned media from the cells or the negative control (media alone). ST is purified human MMP-2 and MMP-9 protein standards which are larger than their murine orthologs. Lower panel shows densitometric quantification of the bands corresponding to MMP-2 and MMP-9 activity. Bar graph is the mean ± SE of density of MMP-2 or MMP-9 from 5 independent experiments. (B) Bar graph showing the effect of re-introduction of CRT in to crt−/− cells (CRT-crt−/−) on MMP-2 and MMP-9 activity. Values are the mean ± SE of MMP-2 or MMP-9 from 4 independent experiments. ⁎P b 0.05 significantly different compared to the wt.
pathways (Lijnen, 2001). Membrane type-MMP (e.g. MT1MMP) is also important for the activation of pro-MMPs (Cowell et al., 1998). In addition to activators of MMP, the cell also secretes MMP inhibitors, e.g. TIMP-1 and TIMP-2 (Lafleur et al., 2003). Strongin and colleagues (Strongin et al., 1995) were the first to report formation of a complex comprising MT1-MMP, TIMP-2 and MMP-2 associated with cell surface. Subsequently, they showed that this tri-molecular complex MT1-MMP/TIMP-2/MMP-2 was involved in pro-MMP-2 activation (Cowell et al., 1998; Lafleur et al., 2003; Zucker et al., 2004). The activities of MMP-2 and MMP-9 are also
modulated by many factors including fibronectin (Esparza et al., 1999), integrin αvβ3 (Hofmann et al., 2000), and thrombospondin-2 (Bornstein et al., 2004). In addition to regulation at the protein level, the activity of MMPs is also modulated at the transcriptional level via many factors such as PI 3-kinase/Akt/ mTOR signalling (Zhang et al., 2004), Raf/ERK pathway (Zhang et al., 2004), MAPK/ERK/JNK pathway (Donnini et al., 2004), Ras/MAPK pathway (Esparza et al., 1999), NF-κB and activator protein-1 (Ogawa et al., 2004), and intracellular Ca2+ levels (Lohi and Keski-Oja, 1995; Mukhopadhyay et al., 2004). Calreticulin (CRT) is an endoplasmic reticulum (ER) calcium binding protein and chaperone which is ubiquitously expressed (Michalak et al., 1999). Targeted deletion of the CRT gene results in embryonic lethality due to a marked decrease in the ventricular wall thickness and deep intertrabecular recesses (Mesaeli et al., 1999; Rauch et al., 2000). The other main defect in the CRT deficient mice is the presence of omphaloceal in older embryos (beyond 16 dpc) (Mesaeli et al., 1999; Rauch et al., 2000). These defects could result from altered extracellular matrix composition. However, no data are available on the role of CRT in regulating MMP activity. Therefore, we tested the hypothesis that the defects observed in the heart and body wall of the CRT deficient embryos are due in part to changes in the MMP-2 and MMP-9 activities. Herein, we show that conditioned media from the CRT deficient (crt−/−) mouse embryonic fibroblast cells had significantly lower MMP-9 activity and increased MMP-2 activity when compared to conditioned media from the wild type (wt) cells. We also demonstrated that the changes in MMP activity correlated to the changes in both the protein and mRNA levels of these genes. Furthermore, we showed that the changes observed in the MMP-2 and MMP-9 mRNA levels in the crt−/− cells are mediated via the PI3 kinase and Ras/Raf/MEK pathway.
Fig. 2. Western blot analysis of MMP-2 and MMP-9 protein levels in wt and crt−/− cells. Cells were cultured in conditioned media for 48 h, followed by lysis in RIPA buffer. Thirty micrograms of protein from the cell lysate or conditioned media was separated on SDS–PAGE. Blots were probed with antibodies to MMP-2 or MMP-9. The blots containing the cell lysates were also probed with an antibody to actin to normalize for loading. Bands corresponding to MMP-2, MMP-9 and actin were quantified by densitometry using the QuantityOne Program. Bar graph shows the mean ± SE of the MMP/actin protein expression from 4 independent experiments. ⁎P b 0.05 significantly different compared to the wt.
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2. Results 2.1. MMP-2 and MMP-9 activity and protein expression To test if there were any changes in the proteases that degrade the extracellular matrix in the absence of CRT, we measured the activity of MMP-2 and MMP-9 in conditioned media from the wt
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and crt−/− cells using gelatin zymography. In the absence of CRT there was a significant decrease in MMP-9 activity and a significant increase in MMP-2 activity in the culture media (Fig. 1), however the net change in the gelatinase activity was significantly reduced in the crt−/− cells as compared to the wt cells. To determine whether the changes in MMP-2 and MMP-9 activities were due to deletion of CRT, crt−/− cells were
Fig. 3. Immunohistochemical staining of wt and crt−/− mouse embryos using antibodies for MMP-2 (A) and MMP-9 (B). Four-micrometer sections of mouse embryos (E14.5) were labelled with polyclonal antibody to MMP-2 (A) or MMP-9 (B) followed by HRP labelled secondary antibody and DAB. Nuclei of the cells were counter stained with haematoxylin. H represents ventricular wall; UA is umbilical artery.
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Fig. 4. Measurement of the mRNA levels of MMP-2 and MMP-9 in the wt and crt−/− cells. Cells were cultured in conditioned media as described in the Experimental procedures, followed by lysis and RNA isolation using Trizol™ reagent. RT-PCR was carried out using primers specific to murine MMP-2, MMP-9 and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9 and GAPDH were quantified by densitometry using the Quantity One Program. Bar graph shows the mean ± SE of the MMP/GAPDH signal from 4 independent experiments. ⁎P b 0.05 significantly different as compared to the wt.
MMP-2 was significantly reduced as compared to the crt−/− cells. Western blot analysis with antibody to MMP-2 showed no significant change in the level of MMP-2 protein level in the cell lysate of the crt−/− cells (Fig. 2). However, MMP-9 protein level was significantly decreased in the total cell lysate prepared from the crt−/− cells as compared to the wt cells (Fig. 2). The mature MMP proteins are usually secreted from the cell where they associate with the cell surface (Strongin et al., 1995). Upon activation MMP is then released into the culture media or the extracellular space of the tissue. Therefore, to assess the changes in the total level of MMP-2 and MMP-9 proteins in the extracellular space we used immunohistochemical staining. Paraffin sections from the 14 day old wt and crt−/− mouse embryos were stained with MMP-2 and MMP-9 specific antibodies followed with DAB. As shown in Fig. 3A, MMP-2 expression is higher in the crt−/− hearts and umbilical vessel wall as compared to the wt tissue. Furthermore, the expression of MMP-9 was significantly lower in the crt−/− tissue as compared to the wt (Fig. 3B). Therefore, the changes in the activity of both MMP-2 and MMP-9 in the absence of CRT reflect the level of protein expression in the affected tissues.
transfected with a mammalian expression plasmid containing CRT cDNA (described in Mesaeli and Phillipson, 2004). As shown in Fig. 1B, CRT-crt−/− cells showed a significant increase in the activity of MMP-9 and at the same time the activity of
Fig. 5. Measurement of the mRNA levels of MT1-MMP (A) and TIMP-1, TIMP-2 (B) in the wt and crt−/− cells. Cells were cultured in conditioned media for 24 h, followed by lysis and RNA isolation. RT-PCR was carried out using primers specific to murine MT1-MMP, TIMP-1, TIMP-2 and GAPDH as listed in Table 1. Bands corresponding to MT1-MMP, TIMP-1, TIMP-2 and GAPDH were quantified by densitometry using the QuantityOne Program. Bar graph shows the mean ± SE of the MT1-MMP (or TIMP-1,-2)/GAPDH signal from 4 different experiments. P b 0.05 significantly different compared to the wt.
Fig. 6. Effect of insulin on MMP-2 and MMP-9 activity in wt and crt−/− cells. Cells were cultured in media containing DMEM alone (C) or the conditioned media (Ins) for 48 h, then the media were collected and zymography was carried out. (A) Bar graph showing densitometric quantification of the bands corresponding to MMP-2 activity. (B) shows quantification of the bands corresponding to MMP-9 activity. Values are the mean ± SE of density of MMP-2 or MMP-9 from 3 independent experiments. ⁎P b 0.05 significantly different compared to C (DMEM).
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2.2. MMP-2 and MMP-9 mRNA level The observed changes in the MMP-2 and MMP-9 protein levels could be due to altered levels of their mRNAs. To address this possibility, we used semi-quantitative RT-PCR to measure the level of mRNA for MMP-2, MMP-9, as well as their modifiers MT1-MMP, TIMP-1 and TIMP-2. As shown in Fig. 4, there was a significant increase in the mRNA expression of MMP-2 accompanied by a significant decrease in the MMP-9 mRNA expression in the crt−/− cells as compared to the wt cells. This increase in MMP-2 mRNA was accompanied by a significant increase in the MT1-MMP mRNA level in the crt−/− cells (Fig. 5A). Furthermore, no changes in the mRNA level of the endogenous inhibitors of MMP-2 and MMP-9, TIMP-2 and TIMP-1 were detected in the crt−/− cells, respectively (Fig. 5B). 2.3. Role of insulin in the MMP-2 and MMP-9 expression and activity in the crt−/− cells The conditioned media in our experiments contained insulin, therefore we first tested whether insulin contributes to the changes in the MMP expression and activity in the crt−/− cells. Fig. 6A shows that the basal activity of MMP-2 is similar in the wt and crt−/− cells. Furthermore, insulin increased this activity in both cell types albeit to a higher level in the crt−/− cells (Fig. 6A). On the other hand, the basal activity of MMP-9 was lower in
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the crt−/− cells, although not statistically significant (Fig. 6B). Similar to MMP-2, addition of insulin to the media resulted in increased MMP-9 activity in both the wt and crt−/− cells (Fig. 6B). Similar changes were also observed in the mRNA of the MMP-2 and MMP-9 (data not shown). Phosphatidylinositol 3 kinase (PI3 kinase) and Ras/Raf/MEK are two downstream signalling pathways of insulin receptor. Thus we examined these two pathways in the regulation of MMP expression in the absence of CRT using inhibitors of these two pathways. As shown in Fig. 7A, wortmannin (inhibitor of PI3 kinase) significantly reduced the level of MMP-2 mRNA in the wt and crt−/− cells. However, the mRNA level of MMP-2 after this treatments was still significantly higher (Fig. 7A) in the treated crt−/− cells than in the treated wt cells. On the other hand, inhibition of PI3 kinase led to a significant decrease in the MMP-9 mRNA (Fig. 7B). Interestingly, blockade of PI3 kinase in the wt cells did not affect the mRNA level of MT1-MMP, while this treatment decreased the MT1-MMP mRNA in the crt−/− cells (Fig. 7C). Indeed, following PI3 kinase inhibition, the level of MT1-MMP in the crt−/− cells was similar to that of wt cells. PI3 kinase inhibition had no effect on the mRNA level of TIMP-1 or -2 (data not shown). Next we examined the effect of insulin and PI3 kinase inhibitor on the activity of MMP-2 and MMP-9 using gelatin zymography. As shown in Fig. 7D, pre-treatment of the cells with wortmannin for 30 min followed by incubation with wortmannin and insulin resulted in suppression of
Fig. 7. Effect of inhibition of PI3 kinase on the mRNA levels of MMP-2 (A), MMP-9 (B) and MT1-MMP (C) in the wt and crt−/− cells. Cells were cultured in conditioned media containing wortmannin (Wort.) and insulin (Ins) followed by lysis and RNA isolation using Trizol™ reagent. RT-PCR was carried out using primers specific to murine MMP-2 (A), MMP-9 (B), MT1-MMP (C) and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9, MT1-MMP (C) and GAPDH were quantified by densitometry using QuantityOne Program. Bar graph shows the mean ± SE of the MMP/GAPDH signal from 4 independent experiments. (D) A representative zymograph showing changes in the activity of MMP-2 and MMP-9 in conditioned media from the wt and crt−/− cells treated with DMEM + 0.2% DMSO, DMEM + 0.2% DMSO + insulin, and wortmannin. †P b 0.05 significantly different compared to the wt. ⁎P b 0.001 significantly different compared to the corresponding untreated cells.
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Fig. 8. Effect of MEK inhibitor on MMP-2 and MMP-9 expression in the wt and crt−/− cells. Cells were cultured in conditioned media containing MEK inhibitor (PD98059, PD) and insulin (Ins). Cells were lysed and RNA was isolated using Trizol™ reagent. Conditioned media were precipitated using trichloroacetic acid as described in Experimental procedures. RT-PCR was carried out using primers specific to murine MMP-2, MMP-9 and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9 and GAPDH were quantified by densitometry using the QuantityOne Program. (A, C) Bar graphs showing the mean ± SE of the MMP/GAPDH signal from 4 different experiments. (B, D) Representative western blot showing changes in the MMP-2 (B) and MMP-9 (D) protein level in the conditioned media following treatment with PD98059. †P b 0.05 significantly different compared to the corresponding wt cells. ⁎P b 0.05 significantly different compared to the corresponding untreated cells.
MMP-2 and MMP-9 activities in both the wt and crt−/− cells as compared to the cells treated with conditioned media containing insulin (with 0.2% DMSO). Finally, the changes in the MMP activities correspond to the MMP-2 and MMP-9 mRNA expression in the wt and crt−/− cells (Fig. 7A, B, D). Inhibition of MEK (using PD98059) caused no change in the MMP-2 mRNA level in the wt (Fig. 8A) while crt−/− cells showed an increase in MMP-2 mRNA although not statistically significant. Fig. 8B shows that the level of MMP-2 protein secreted in the conditioned media also did not change significantly following PD98059 treatment. Intriguingly, inhibition of the MEK pathway by PD98059 and insulin resulted in a significant increase in the MMP-9 mRNA of the crt−/− cells with no change in the MMP-9 mRNA in the wt cells (Fig. 8C). The changes in the level of MMP-9 protein level in the conditioned media following PD98059 reflected that of MMP-9 mRNA (Fig. 8D). These data suggest that in the crt−/− cells insulin mediated activation of the Ras/Raf/MEK pathway inhibits the expression of MMP-9. At the same time, insulin's effect on activation of PI3 kinase elicits a significant increase in MMP-2 expression in the crt−/− cells. 3. Discussion Targeted deletion of the CRT gene has been shown to result in the thinning of the ventricular wall, increased intertrabecular recesses and the presence of omphaloceal in mouse embryos
(Mesaeli et al., 1999; Rauch et al., 2000). The failure of resorption of umbilical hernia (omphaloceal) implies that there are defects in wound healing process in the absence of CRT. Rauch et al. (2000) reported that ES derived crt−/− neuronal cells had defects in the wound healing process. Collectively, these defects could be due to altered extracellular matrix composition in the absence of CRT. However, to date no data are available on the effect of CRT in altering ECM composition or its degradation. MMP-2 and MMP-9 are two gelatinases which have been shown to regulate cardiac and vascular remodelling (Lijnen, 2001) and they have also been implicated in the regulation of wound healing (Simeon et al., 1999). In this study we showed, for the first time, a differential expression and activity of MMP-2 and MMP-9 in CRT deficient cells ((Figs. 1, 2, 4)). We have also demonstrated that partial restoration of CRT expression in the crt−/− cells could reverse the activities of MMP-2 and MMP-9 (Fig. 1B). Our data illustrate that these changes were not due to altered expression of endogenous inhibitors of MMP-2 and MMP-9 (TIMP-1 and TIMP-2, Fig. 5), but were due in part to altered PI3 kinase (Fig. 7) and/or MEK kinase activity (Fig. 8). MMP-2 and MMP-9 are both gelatinases which share a large number of extracellular matrix substrates (Nagase and Woessner, 1999), thus one might expect that the increase in MMP-2 activity and expression could compensate for the loss of MMP-9 activity in CRT deficient embryos and cells. However, our data show that in CRT deficient cells the elevated activity of MMP-2 is
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significantly lower than the attenuation of MMP-9 activity (Fig. 1). Overall, the additive effect of these changes will result in a significant loss of gelatinolytic activity in the crt−/− mice therefore it could contribute to the observed defects in the heart and omphaloceal formation in the crt−/− embryos (Mesaeli et al., 1999; Rauch et al., 2000). CRT is a molecular chaperone regulating the proper folding of many proteins in the ER (Ellgaard and Frickel, 2003; Guo et al., 2003). CRT is also involved in the regulation of intracellular Ca2+ homeostasis (Mery et al., 1996; Michalak et al., 1999) thus affecting expression of many genes at the transcriptional level (Waser et al., 1997). The decreased activity of MMP-9 in the absence of CRT (Fig. 1) therefore suggests a defect in MMP-9 protein synthesis or folding. However, we showed that these changes reflect a decrease in MMP-9 expression at the level of both the protein (Figs. 2 and 3) and mRNA (Fig. 4) in the crt−/− cells. On the other hand, cellular content of MMP-2 protein was not different between the crt−/− and wt cells, which could be due to shedding of the activated MMP-2 from the cell surface as was reported previously (Strongin et al., 1995). Increased expression of MT1-MMP has also been shown to activate proteolysis of pro-MMP-2 and release of the active MMP-2 in the extracellular space (Hofmann et al., 2000; Zhang and Brodt, 2003). Therefore, our observation on increased activity of MMP-2 in the crt−/− cells could be due to a combination of both increased MMP-2 expression and MT1-MMP expression and activity (Figs. 4 and 5). Two endogenously expressed inhibitory proteins TIMP-2 and TIMP-1 are also important in the regulation of MMP-2 and MMP-9 activity, respectively (Lafleur et al., 2003; Nagase and Woessner, 1999). In this study, we showed no changes in the expression of these two proteins in the crt−/− cells as compared to the wt cells (Fig. 5B). Therefore, the decreased activity of MMP-9 and increased MMP-2 activity are not due to changes in their inhibitory proteins. Furthermore, our results on corresponding changes in the protein and mRNA level of MMP-2 and MMP-9 in the crt−/− cells (Fig. 4) suggest that the effect of CRT on MMPs is not via its chaperone property but rather via transcriptional regulation of these genes. Previous studies showed that insulin and IGF-1 up-regulate MMP-2 expression in different cell types (Yoon and Hurta, 2001; Zhang et al., 2004), and this effect was due to both PI3 kinase dependent and independent pathways (Shin et al., 2005; Ulku et al., 2003; Yoon and Hurta, 2001). IGF receptor stimulation has also been shown to increase MT1-MMP activation in the Lewis lung carcinoma cell line (Zhang and Brodt, 2003). However, the role of insulin or IGF in the regulation of MMP-9 expression is not clear. In addition, the effect of the PI3 kinase activation on MMP-9 expression seems to be dependent on cell type. Glial derived neurotropic peptide has been shown to increase MMP-9 expression via a PI3-kinase dependent mechanism in MIA PaCa-2 cells (Okada et al., 2003). Whereas, PI3 kinase activation via the PDGF receptor was shown to inhibit cytokine (TNFα and IL-1) mediated increase in MMP-9 expression in C6 glioma cell line (Esparza et al., 1999). Recently, we have observed a significant increase in insulin receptor density and activation of the PI3 kinase/Akt activity in
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the crt−/− cells following insulin stimulation (Jalali and Mesaeli, manuscript under revision). Therefore, these changes in insulin receptor activity could in part explain the increased MMP-2 expression. This effect of insulin receptor/PI3 kinase could be at the level of both regulating the MMP-2 mRNA expression and activation of MT1-MMP which activates MMP-2 enzyme. A previous report has shown similar changes in the MMP-2 and MT1-MMP following stimulation of IGF receptors (Zhang and Brodt, 2003). Indeed, we showed that inhibition of PI3 kinase by wortmannin resulted in a significant inhibition of MMP-2 and MMP-9 expression (Fig. 7A, B) and activity (Fig. 7D) in the wt and crt−/− cells. However, the decrease in the MMP-2 expression was still significantly higher in the wortmannin treated crt−/− cells compared to the wortmannin treated wt cells. A role for insulin mediated activation of Ras/Raf/MEK in the modulation of MMP-2 and MMP-9 activities has also been reported previously (Shin et al., 2005; Yoon and Hurta, 2001). Indeed, here we have reported a significant increase in MMP-9 mRNA (Fig. 8C) and protein (Fig. 8D) in the crt−/− cells following treatment with MEK inhibitor PD98059. Furthermore, this treatment had no significant effect on the mRNA level of MMP-2 or MMP-9 in the wt cells (Fig. 8). These data suggest involvement of Ras/Raf/MEK pathway in the regulation of MMP-9 gene in the absence of CRT. To date, no data are available on the effect of CRT on the regulation of Ras/Raf/MEK pathway. Our current report is the only study suggesting alteration in these pathways in the absence of CRT. In addition to these two pathways other regulatory pathways such as NFκB (Knipp et al., 2004; Ogawa et al., 2004), intracellular calcium (Lohi and KeskiOja, 1995; Mukhopadhyay et al., 2004), and nitric oxide (MarcetPalacios et al., 2003) have been shown to regulate the expression of MMP-2 and MMP-9. Further investigations are needed to examine the role of these pathways in the regulation of MMP-2 and MMP-9 expression upon loss of CRT function. In conclusion, we showed that targeted deletion of CRT gene results in up-regulation of MMP-2 and MT1-MMP expression and activity accompanied by decreased MMP-9 expression and activity. These changes were partially due to activation of PI3 kinase dependent pathways and alteration of the Ras/Raf/MEK activities. 4. Experimental procedures 4.1. Materials Wortmannin, SB203580 and PD98059 were purchased from Calbiochem (EMD Biosciences Inc., USA). MMP-9 and actin antibodies were purchased from SigmaAldrich (St. Louis, USA). MMP-2 antibody was from MEDICORP (Montreal, QB, Canada), and DAB and ABC kit was from Vector Laboratories (Burlington, ON, Canada). Cell culture media and insulin–transferrin were from Invitrogen (Canada). 4.2. Cell culture and treatment Mouse embryonic fibroblast cells (MEF) from wild type (wt) and calreticulin null (crt−/−) 14-day-old mouse embryos were
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prepared as described previously (Mesaeli and Phillipson, 2004; Mesaeli et al., 1999). For experiments described below, cells were seeded at a density of 2 × 106 cells per plate and cultured in DMEM with 10% FBS at 37 °C. The next day, the cells were washed with PBS and incubated in conditioned media containing DMEM and 1% insulin–transferrin–selenium-A (Invitrogen) for 48 h. To examine the effect of reversal of CRT expression on MMP activities, crt−/− cells were transiently transfected with 10 μg of a plasmid encoding CRT-HA [described in Mesaeli and Phillipson, 2004] or an empty parental plasmid using Lipofectamine 2000. After 24 h the cells were washed and conditioned media were added and zymography experiments were carried out as described below. To determine the effect of different kinase pathways on the MMP-2 and MMP-9 mRNA expression, cells were cultured in conditioned media (DMEM containing 1% insulin–transferrin– selenium-A) for 24 h. The cells were washed with insulin free media and pre-incubated in the presence of specific inhibitors (10 nM PI3 kinase inhibitor wortmannin, or 50 μM MEK inhibitor PD98059 dissolved in DMSO) in insulin free media for 30 min. Next, the media was changed to DMEM containing the specific inhibitor and insulin–transferrin–selenium-A. In some experiments 0.2% DMSO was added to DMEM to examine the effect of the DMSO vehicle on MMP-2 and MMP-9 activities. The treated cells were then used to isolate RNA as described below. The conditioned media from these cells were used for examining the level of MMP-2 and MMP-9 as described below. 4.3. Zymography Conditioned media from the treated cells, non-treated cells and plates containing no cells (media control) were harvested and centrifuged at 2000 rpm for 10 min at room temperature. The collected media were then used for zymography. Twenty microliters of conditioned media was mixed with sample buffer containing 20% glycerol, 4% SDS, 0.13 M Tris, pH 6.8 and resolved on a 7.5% polyacrylamide gel containing 1 mg/ml porcine gelatin (Sigma). After electrophoresis, the gels were washed twice with 2.5% TritonX-100 for 30 min at room temperature to remove the SDS, followed by incubation in a buffer containing; 50 mM Tris–HCl, 5 mM CaCl2, 0.2 M NaCl, pH 7.6, at 37 °C for 48 h. The gels were then stained in Coomassie blue R-250 for 30 min at room temperature and destained with 40% methanol and 10% acetic acid over night at room temperature. Clear zones against the blue background indicated gelatinase activity. Purified human MMP-2 and MMP-9 (Chemicon International) were used as markers for zymography gels. To quantify the gelatinase activity, the stained zymography gels were scanned and the density of clear bands was calculated using the BioRad QuantityOne program. 4.4. Immunohistochemistry Paraffin sections (4 μm) from wt and crt−/− mouse embryos were used for immunohistochemistry using the ABC Kit (Vector Laboratories Inc.). Embryo sections were incubated
with rabbit anti-MMP-2 (1:200), or rabbit anti-MMP-9 (1:300) for 1 h at room temperature, followed by incubation with biotinylated anti-rabbit secondary antibody for 1 h at room temperature. The sections were stained with DAB (diaminobenzidine, Sigma) and nuclei were counter stained with haematoxylin QS (Vector Laboratories Inc.). Images were taken using a Zeiss Axioscop microscope using Axiovision software. 4.5. Western blot Cell were lysed using RIPA buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 0.5% NaDOC, 0.1% SDS, 0.15 M NaCl, pH 7.5) containing phenylmethysulfonyl fluoride (PMSF) and a cocktail of protease inhibitors (Sigma) (Mesaeli et al., 1999). Subsequently, the cell lysates were centrifugated at 7000 rpm, 4 °C for 5 min. The protein concentration of the supernatant was determined by a BioRad protein assay kit, using BSA as a standard. 10–50 μg protein was separated on 7.5% SDS– acrylamide gel. Western blot was performed using an antibody to MMP-2 or MMP-9 (Sigma) and Supersignal Chemiluminescent Substrate. Bands corresponding to MMP-2 and MMP-9 were quantified using QuantityOne program (BioRad). The level of MMP-2 and MMP-9 in conditioned media was analyzed following trichloroacetic acid precipitation. Briefly, after treatment of the cells with different inhibitors the total volume of the conditioned media mixed with 7.5% (v/v) trichloroacetic acid and 0.02% (v/v) sodium deoxycholate solutions. The mixture was incubated at 4 °C for 1 h followed by centrifugation at 13,200 rpm for 10 min at 4 °C. Protein pellets were washed several times with ice cold acetone, dried and re-suspended in Laemmli buffer. The proteins were then resolved on 7.5% SDS–acrylamide gel followed by western blot with MMP-2 and MMP-9 antibodies. 4.6. Reverse transcriptase assay Total RNA was isolated from the wt and crt−/− cells using Trizol™ reagent (Invitrogen). SuperScript™ II RT and Poly-dT oligo were used to generate cDNA from the RNA. MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2 and GAPDH were amplified using the Platinum Taq DNA polymerase (Invitrogen)
Table 1 List of primers used for RT-PCR Primer MMP-2 MMP-9 MT1-MMP TIMP-1 TIMP-2 GAPDH
Sequence Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense
5′-CATCGCCCATCATCAAGTTCC-3′ 5′-CCGAGCAAAAGCATCATCCAC-3′ 5′-TGATGCCGGCAGACAATCC-3′ 5′-CCTTATCCACGCGAATGACG-3′ 5′-ATGAAGAATTCAGGGCAGTGG-3′ 5′-GACCTTGTCCAGCAGCGAACGC-3′ 5′-TAAGGCCTGTAGCTGTGCC-3′ 5′-TCTTCATGCGGTTCTGGG-3′ 5′-TTTGCAATGCAGACGTAGTG-3′ 5′-TTCCTCCAACGTCCAGCG-3′ 5′-GCCAAGGTCATCCATGACAAC-3′ 5′-GTCCACCACCCTGTTGCTGTA-3′
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and gene specific primers listed in Table 1. In preliminary experiments the conditions of RT-PCR were optimized to determine the cycle number and annealing temperatures required for each primer set to obtain best results. Equal aliquots of the PCR reaction were resolved on a 1.5% agarose gel and bands corresponding to each gene listed above and GAPDH of the same sample were quantified using the BioRad QuantityOne Program. The data were then presented as the ratio of signal normalized to GAPDH signal (mean ± SE). Data were analyzed using the Student's t-test. Acknowledgment We thank Mr. Michael Wiwchar for superb technical assistance. This work was supported by a grant to N.M. from the Canadian Institute of Health Research (CIHR MOP# 42424). M.W. was the recipient of a fellowship from the University of Manitoba, H.M. is a Postdoctoral fellow of Canadian Institute of Health Research. N.M. is a new Investigator of Heart and Stroke Foundation of Canada. References Bornstein, P., Agah, A., Kyriakides, T.R., 2004. The role of thrombospondins 1 and 2 in the regulation of cell–matrix interactions, collagen fibril formation, and the response to injury. Int. J. Biochem. Cell Biol. 36, 1115–1125. Cowell, S., Knauper, V., Stewart, M.L., D'Ortho, M.P., Stanton, H., Hembry, R.M., Lopez-Otin, C., Reynolds, J.J., Murphy, G., 1998. Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3. Biochem. J. 331 (Pt 2), 453– 458. Creemers, E.E., Cleutjens, J.P., Smits, J.F., Daemen, M.J., 2001. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ. Res. 89, 201–210. Donnini, S., Morbidelli, L., Taraboletti, G., Ziche, M., 2004. ERK1–2 and p38 MAPK regulate MMP/TIMP balance and function in response to thrombospondin-1 fragments in the microvascular endothelium. Life Sci. 74, 2975–2985. Egeblad, M., Werb, Z., 2002. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev., Cancer 2, 161–174. Ellgaard, L., Frickel, E.M., 2003. Calnexin, calreticulin, and ERp57: teammates in glycoprotein folding. Cell Biochem. Biophys. 39, 223–247. Esparza, J., Vilardell, C., Calvo, J., Juan, M., Vives, J., Urbano-Marquez, A., Yague, J., Cid, M.C., 1999. Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways. Blood 94, 2754–2766. Guo, L., Groenendyk, J., Papp, S., Dabrowska, M., Knoblach, B., Kay, C., Parker, J.M., Opas, M., Michalak, M., 2003. Identification of an N-domain histidine essential for chaperone function in calreticulin. J. Biol. Chem. 278, 50645–50653. Hofmann, U.B., Westphal, J.R., Van Kraats, A.A., Ruiter, D.J., Van Muijen, G.N., 2000. Expression of integrin alpha(v)beta(3) correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo. Int. J. Cancer 87, 12–19. Knipp, B.S., Ailawadi, G., Ford, J.W., Peterson, D.A., Eagleton, M.J., Roelofs, K.J., Hannawa, K.K., Deogracias, M.P., Ji, B., Logsdon, C., Graziano, K.D., Simeone, D.M., Thompson, R.W., Henke, P.K., Stanley, J.C., Upchurch Jr., G.R., 2004. Increased MMP-9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-kappaB and activator protein-1 activity. J. Surg. Res. 116, 70–80.
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Lafleur, M.A., Tester, A.M., Thompson, E.W., 2003. Selective involvement of TIMP-2 in the second activational cleavage of pro-MMP-2: refinement of the pro-MMP-2 activation mechanism. FEBS Lett. 553, 457–463. Li, H., Simon, H., Bocan, T.M., Peterson, J.T., 2000. MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE- and MMPinhibition. Cardiovasc. Res. 46, 298–306. Lijnen, H.R., 2001. Role of the fibrinolytic and matrix metalloproteinase systems in arterial neointima formation after vascular injury. Verh. K. Acad. Geneeskd. Belg. 63, 605–622. Lohi, J., Keski-Oja, J., 1995. Calcium ionophores decrease pericellular gelatinolytic activity via inhibition of 92-kDa gelatinase expression and decrease of 72-kDa gelatinase activation. J. Biol. Chem. 270, 17602–17609. Marcet-Palacios, M., Graham, K., Cass, C., Befus, A.D., Mayers, I., Radomski, M.W., 2003. Nitric oxide and cyclic GMP increase the expression of matrix metalloproteinase-9 in vascular smooth muscle. J. Pharmacol. Exp. Ther. 307, 429–436. McCawley, L.J., Matrisian, L.M., 2000. Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol. Med. Today 6, 149–156. Mery, L., Mesaeli, N., Michalak, M., Opas, M., Lew, D.P., Krause, K.H., 1996. Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx. J. Biol. Chem. 271, 9332–9339. Mesaeli, N., Phillipson, C., 2004. Impaired p53 expression, function, and nuclear localization in calreticulin-deficient cells. Mol. Biol. Cell 15, 1862–1870. Mesaeli, N., Nakamura, K., Zvaritch, E., Dickie, P., Dziak, E., Krause, K.H., Opas, M., MacLennan, D.H., Michalak, M., 1999. Calreticulin is essential for cardiac development. J. Cell Biol. 144, 857–868. Michalak, M., Corbett, E.F., Mesaeli, N., Nakamura, K., Opas, M., 1999. Calreticulin: one protein, one gene, many functions. Biochem. J. 344 (Pt 2), 281–292. Mukhopadhyay, S., Munshi, H.G., Kambhampati, S., Sassano, A., Platanias, L.C., Stack, M.S., 2004. Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma. J. Biol. Chem. 279, 33139–33146. Nagase, H., Woessner Jr., J.F., 1999. Matrix metalloproteinases. J. Biol. Chem. 274, 21491–21494. Ogawa, K., Chen, F., Kuang, C., Chen, Y., 2004. Suppression of matrix metalloproteinase-9 transcription by transforming growth factor-beta is mediated by a nuclear factor-kappaB site. Biochem. J. 381, 413–422. Okada, Y., Eibl, G., Duffy, J.P., Reber, H.A., Hines, O.J., 2003. Glial cellderived neurotrophic factor upregulates the expression and activation of matrix metalloproteinase-9 in human pancreatic cancer. Surgery 134, 293–299. Pyo, R., Lee, J.K., Shipley, J.M., Curci, J.A., Mao, D., Ziporin, S.J., Ennis, T.L., Shapiro, S.D., Senior, R.M., Thompson, R.W., 2000. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J. Clin. Invest. 105, 1641–1649. Rauch, F., Prud'homme, J., Arabian, A., Dedhar, S., St-Arnaud, R., 2000. Heart, brain, and body wall defects in mice lacking calreticulin. Exp. Cell Res. 256, 105–111. Shin, I., Kim, S., Song, H., Kim, H.R., Moon, A., 2005. H-ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells. J. Biol. Chem. 280, 14675–14683. Simeon, A., Monier, F., Emonard, H., Gillery, P., Birembaut, P., Hornebeck, W., Maquart, F.X., 1999. Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide–copper complex glycyl-L-histidyl2+ L-lysine-Cu . J. Invest. Dermatol. 112, 957–964. Spinale, F.G., Coker, M.L., Heung, L.J., Bond, B.R., Gunasinghe, H.R., Etoh, T., Goldberg, A.T., Zellner, J.L., Crumbley, A.J., 2000. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 102, 1944–1949. Strongin, A.Y., Collier, I., Bannikov, G., Marmer, B.L., Grant, G.A., Goldberg, G.I., 1995. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J. Biol. Chem. 270, 5331–5338.
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Ulku, A.S., Schafer, R., Der, C.J., 2003. Essential role of Raf in Ras transformation and deregulation of matrix metalloproteinase expression in ovarian epithelial cells. Mol. Cancer. Res. 1, 1077–1088. Waser, M., Mesaeli, N., Spencer, C., Michalak, M., 1997. Regulation of calreticulin gene expression by calcium. J. Cell Biol. 138, 547–557. Yoon, A., Hurta, R.A., 2001. Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells. Mol. Cell. Biochem. 223, 1–6. Zhang, D., Brodt, P., 2003. Type 1 insulin-like growth factor regulates MT1MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling. Oncogene 22, 974–982.
Zhang, D., Bar-Eli, M., Meloche, S., Brodt, P., 2004. Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals. J. Biol. Chem. 279, 19683–19690. Zucker, S., Hymowitz, M., Conner, C., DeClerck, Y., Cao, J., 2004. TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface. Exp. Cell Res. 293, 164–174.
Differential expression and activity of matrix metalloproteinase-2 and -9 in the calreticulin deficient cells Min Wu a,1 , Hamid Massaeli b,2 , Melanie Durston a , Nasrin Mesaeli a,⁎ a
Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, Department of Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada b Institute of Clinical Research of Montréal, Montreal, Canada Received 28 February 2006; received in revised form 6 February 2007; accepted 23 February 2007
Abstract Calreticulin is an endoplasmic reticulum protein important in cardiovascular development. Deletion of the calreticulin gene leads to defects in the heart and the formation of omphaloceal. These defects could both be due to changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of the MMPs which are essential for cardiovascular remodelling and development. Here, we tested the hypothesis that the defects observed in the heart and body wall of the calreticulin null embryos are due to alterations in MMP-2 and MMP-9 activity. Our results demonstrate that there is a significant decrease in the MMP-9 and increase in the MMP-2 activity and expression in the calreticulin deficient cells. We also showed that there is a significant increase in the expression level of membrane type-1 matrix metalloproteinase (MT1-MMP). In contrast, there was no change in the tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2 in the calreticulin deficient cells as compared to the wild type cells. Interestingly, the inhibition of the MEK kinase pathway using PD98059 attenuated the decrease in the MMP-9 mRNA with no effect on the MMP-2 mRNA level in the calreticulin deficient cells. Furthermore, PI3 kinase inhibitor decreased the expression of both the MMP-2 and MMP-9. This study is the first report on the role of calreticulin in regulating MMP activity. © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Calreticulin; Matrix metalloproteinase-2; MMP-9; MT1-MMP; PI3 kinase; MEK
1. Introduction Extracellular matrix (ECM) is an important structure within tissues that provides a supportive environment for cellular growth, development and morphogenesis. Changes in the ECM
Abbreviations: MMP, matrix metalloproteinase; CRT, calreticulin, crt−/−, calreticulin deficient; wt, wild type; MT1-MMP, membrane type-1 matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; ECM, extracellular matrix; PI3 kinase, phosphatidylinositol 3 kinase. ⁎ Corresponding author. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Tel.: +1 204 235 3671; fax: +1 204 231 1151. E-mail address: [email protected] (N. Mesaeli). 1 Current address: The cardiovascular department of the first affiliated hospital of Medical College of Shantou University, Shantou, Guangdong, 515041, the People's Republic of China. 2 Current address: Institute of Cardiovascular Sciences, St. Boniface Research Centre, Winnipeg, Manitoba, Canada.
composition are important steps associated with the processes of embryogenesis, tissue remodelling, cancer cell invasion, metastasis and angiogenesis (Egeblad and Werb, 2002; McCawley and Matrisian, 2000). Matrix metalloproteinases (MMPs) are a superfamily of zinc-binding endopeptidases that degrade the ECM. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are two members of MMPs which degrade native collagen type IV, collagen type V, fibronectin, entactin, and elastin (Nagase and Woessner, 1999). They play an important role in many processes which lead to cardiovascular disease such as vascular remodelling (Li et al., 2000), atherosclerotic plaque instability, heart failure (Spinale et al., 2000), development of aneurysms (Pyo et al., 2000), myocardial infarction, and left ventricular remodelling after myocardial infarction (Creemers et al., 2001). MMP-2 and MMP-9 are synthesized as pro-enzymes which are activated upon proteolysis by different mechanisms including both plasmin-independent and plasmin-dependent
0945-053X/$ - see front matter © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2007.02.005
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Fig. 1. Analysis of MMP-2 and MMP-9 activity in wild type (wt) and CRT deficient (crt−/−) mouse embryonic fibroblast cells (MEF) using gelatin zymography. Cells were cultured in conditioned media for 48 h, then the media were collected and zymography was carried out (as described in Experimental procedures). (A) Upper panel shows a representative zymography of the conditioned media from the cells or the negative control (media alone). ST is purified human MMP-2 and MMP-9 protein standards which are larger than their murine orthologs. Lower panel shows densitometric quantification of the bands corresponding to MMP-2 and MMP-9 activity. Bar graph is the mean ± SE of density of MMP-2 or MMP-9 from 5 independent experiments. (B) Bar graph showing the effect of re-introduction of CRT in to crt−/− cells (CRT-crt−/−) on MMP-2 and MMP-9 activity. Values are the mean ± SE of MMP-2 or MMP-9 from 4 independent experiments. ⁎P b 0.05 significantly different compared to the wt.
pathways (Lijnen, 2001). Membrane type-MMP (e.g. MT1MMP) is also important for the activation of pro-MMPs (Cowell et al., 1998). In addition to activators of MMP, the cell also secretes MMP inhibitors, e.g. TIMP-1 and TIMP-2 (Lafleur et al., 2003). Strongin and colleagues (Strongin et al., 1995) were the first to report formation of a complex comprising MT1-MMP, TIMP-2 and MMP-2 associated with cell surface. Subsequently, they showed that this tri-molecular complex MT1-MMP/TIMP-2/MMP-2 was involved in pro-MMP-2 activation (Cowell et al., 1998; Lafleur et al., 2003; Zucker et al., 2004). The activities of MMP-2 and MMP-9 are also
modulated by many factors including fibronectin (Esparza et al., 1999), integrin αvβ3 (Hofmann et al., 2000), and thrombospondin-2 (Bornstein et al., 2004). In addition to regulation at the protein level, the activity of MMPs is also modulated at the transcriptional level via many factors such as PI 3-kinase/Akt/ mTOR signalling (Zhang et al., 2004), Raf/ERK pathway (Zhang et al., 2004), MAPK/ERK/JNK pathway (Donnini et al., 2004), Ras/MAPK pathway (Esparza et al., 1999), NF-κB and activator protein-1 (Ogawa et al., 2004), and intracellular Ca2+ levels (Lohi and Keski-Oja, 1995; Mukhopadhyay et al., 2004). Calreticulin (CRT) is an endoplasmic reticulum (ER) calcium binding protein and chaperone which is ubiquitously expressed (Michalak et al., 1999). Targeted deletion of the CRT gene results in embryonic lethality due to a marked decrease in the ventricular wall thickness and deep intertrabecular recesses (Mesaeli et al., 1999; Rauch et al., 2000). The other main defect in the CRT deficient mice is the presence of omphaloceal in older embryos (beyond 16 dpc) (Mesaeli et al., 1999; Rauch et al., 2000). These defects could result from altered extracellular matrix composition. However, no data are available on the role of CRT in regulating MMP activity. Therefore, we tested the hypothesis that the defects observed in the heart and body wall of the CRT deficient embryos are due in part to changes in the MMP-2 and MMP-9 activities. Herein, we show that conditioned media from the CRT deficient (crt−/−) mouse embryonic fibroblast cells had significantly lower MMP-9 activity and increased MMP-2 activity when compared to conditioned media from the wild type (wt) cells. We also demonstrated that the changes in MMP activity correlated to the changes in both the protein and mRNA levels of these genes. Furthermore, we showed that the changes observed in the MMP-2 and MMP-9 mRNA levels in the crt−/− cells are mediated via the PI3 kinase and Ras/Raf/MEK pathway.
Fig. 2. Western blot analysis of MMP-2 and MMP-9 protein levels in wt and crt−/− cells. Cells were cultured in conditioned media for 48 h, followed by lysis in RIPA buffer. Thirty micrograms of protein from the cell lysate or conditioned media was separated on SDS–PAGE. Blots were probed with antibodies to MMP-2 or MMP-9. The blots containing the cell lysates were also probed with an antibody to actin to normalize for loading. Bands corresponding to MMP-2, MMP-9 and actin were quantified by densitometry using the QuantityOne Program. Bar graph shows the mean ± SE of the MMP/actin protein expression from 4 independent experiments. ⁎P b 0.05 significantly different compared to the wt.
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2. Results 2.1. MMP-2 and MMP-9 activity and protein expression To test if there were any changes in the proteases that degrade the extracellular matrix in the absence of CRT, we measured the activity of MMP-2 and MMP-9 in conditioned media from the wt
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and crt−/− cells using gelatin zymography. In the absence of CRT there was a significant decrease in MMP-9 activity and a significant increase in MMP-2 activity in the culture media (Fig. 1), however the net change in the gelatinase activity was significantly reduced in the crt−/− cells as compared to the wt cells. To determine whether the changes in MMP-2 and MMP-9 activities were due to deletion of CRT, crt−/− cells were
Fig. 3. Immunohistochemical staining of wt and crt−/− mouse embryos using antibodies for MMP-2 (A) and MMP-9 (B). Four-micrometer sections of mouse embryos (E14.5) were labelled with polyclonal antibody to MMP-2 (A) or MMP-9 (B) followed by HRP labelled secondary antibody and DAB. Nuclei of the cells were counter stained with haematoxylin. H represents ventricular wall; UA is umbilical artery.
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Fig. 4. Measurement of the mRNA levels of MMP-2 and MMP-9 in the wt and crt−/− cells. Cells were cultured in conditioned media as described in the Experimental procedures, followed by lysis and RNA isolation using Trizol™ reagent. RT-PCR was carried out using primers specific to murine MMP-2, MMP-9 and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9 and GAPDH were quantified by densitometry using the Quantity One Program. Bar graph shows the mean ± SE of the MMP/GAPDH signal from 4 independent experiments. ⁎P b 0.05 significantly different as compared to the wt.
MMP-2 was significantly reduced as compared to the crt−/− cells. Western blot analysis with antibody to MMP-2 showed no significant change in the level of MMP-2 protein level in the cell lysate of the crt−/− cells (Fig. 2). However, MMP-9 protein level was significantly decreased in the total cell lysate prepared from the crt−/− cells as compared to the wt cells (Fig. 2). The mature MMP proteins are usually secreted from the cell where they associate with the cell surface (Strongin et al., 1995). Upon activation MMP is then released into the culture media or the extracellular space of the tissue. Therefore, to assess the changes in the total level of MMP-2 and MMP-9 proteins in the extracellular space we used immunohistochemical staining. Paraffin sections from the 14 day old wt and crt−/− mouse embryos were stained with MMP-2 and MMP-9 specific antibodies followed with DAB. As shown in Fig. 3A, MMP-2 expression is higher in the crt−/− hearts and umbilical vessel wall as compared to the wt tissue. Furthermore, the expression of MMP-9 was significantly lower in the crt−/− tissue as compared to the wt (Fig. 3B). Therefore, the changes in the activity of both MMP-2 and MMP-9 in the absence of CRT reflect the level of protein expression in the affected tissues.
transfected with a mammalian expression plasmid containing CRT cDNA (described in Mesaeli and Phillipson, 2004). As shown in Fig. 1B, CRT-crt−/− cells showed a significant increase in the activity of MMP-9 and at the same time the activity of
Fig. 5. Measurement of the mRNA levels of MT1-MMP (A) and TIMP-1, TIMP-2 (B) in the wt and crt−/− cells. Cells were cultured in conditioned media for 24 h, followed by lysis and RNA isolation. RT-PCR was carried out using primers specific to murine MT1-MMP, TIMP-1, TIMP-2 and GAPDH as listed in Table 1. Bands corresponding to MT1-MMP, TIMP-1, TIMP-2 and GAPDH were quantified by densitometry using the QuantityOne Program. Bar graph shows the mean ± SE of the MT1-MMP (or TIMP-1,-2)/GAPDH signal from 4 different experiments. P b 0.05 significantly different compared to the wt.
Fig. 6. Effect of insulin on MMP-2 and MMP-9 activity in wt and crt−/− cells. Cells were cultured in media containing DMEM alone (C) or the conditioned media (Ins) for 48 h, then the media were collected and zymography was carried out. (A) Bar graph showing densitometric quantification of the bands corresponding to MMP-2 activity. (B) shows quantification of the bands corresponding to MMP-9 activity. Values are the mean ± SE of density of MMP-2 or MMP-9 from 3 independent experiments. ⁎P b 0.05 significantly different compared to C (DMEM).
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2.2. MMP-2 and MMP-9 mRNA level The observed changes in the MMP-2 and MMP-9 protein levels could be due to altered levels of their mRNAs. To address this possibility, we used semi-quantitative RT-PCR to measure the level of mRNA for MMP-2, MMP-9, as well as their modifiers MT1-MMP, TIMP-1 and TIMP-2. As shown in Fig. 4, there was a significant increase in the mRNA expression of MMP-2 accompanied by a significant decrease in the MMP-9 mRNA expression in the crt−/− cells as compared to the wt cells. This increase in MMP-2 mRNA was accompanied by a significant increase in the MT1-MMP mRNA level in the crt−/− cells (Fig. 5A). Furthermore, no changes in the mRNA level of the endogenous inhibitors of MMP-2 and MMP-9, TIMP-2 and TIMP-1 were detected in the crt−/− cells, respectively (Fig. 5B). 2.3. Role of insulin in the MMP-2 and MMP-9 expression and activity in the crt−/− cells The conditioned media in our experiments contained insulin, therefore we first tested whether insulin contributes to the changes in the MMP expression and activity in the crt−/− cells. Fig. 6A shows that the basal activity of MMP-2 is similar in the wt and crt−/− cells. Furthermore, insulin increased this activity in both cell types albeit to a higher level in the crt−/− cells (Fig. 6A). On the other hand, the basal activity of MMP-9 was lower in
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the crt−/− cells, although not statistically significant (Fig. 6B). Similar to MMP-2, addition of insulin to the media resulted in increased MMP-9 activity in both the wt and crt−/− cells (Fig. 6B). Similar changes were also observed in the mRNA of the MMP-2 and MMP-9 (data not shown). Phosphatidylinositol 3 kinase (PI3 kinase) and Ras/Raf/MEK are two downstream signalling pathways of insulin receptor. Thus we examined these two pathways in the regulation of MMP expression in the absence of CRT using inhibitors of these two pathways. As shown in Fig. 7A, wortmannin (inhibitor of PI3 kinase) significantly reduced the level of MMP-2 mRNA in the wt and crt−/− cells. However, the mRNA level of MMP-2 after this treatments was still significantly higher (Fig. 7A) in the treated crt−/− cells than in the treated wt cells. On the other hand, inhibition of PI3 kinase led to a significant decrease in the MMP-9 mRNA (Fig. 7B). Interestingly, blockade of PI3 kinase in the wt cells did not affect the mRNA level of MT1-MMP, while this treatment decreased the MT1-MMP mRNA in the crt−/− cells (Fig. 7C). Indeed, following PI3 kinase inhibition, the level of MT1-MMP in the crt−/− cells was similar to that of wt cells. PI3 kinase inhibition had no effect on the mRNA level of TIMP-1 or -2 (data not shown). Next we examined the effect of insulin and PI3 kinase inhibitor on the activity of MMP-2 and MMP-9 using gelatin zymography. As shown in Fig. 7D, pre-treatment of the cells with wortmannin for 30 min followed by incubation with wortmannin and insulin resulted in suppression of
Fig. 7. Effect of inhibition of PI3 kinase on the mRNA levels of MMP-2 (A), MMP-9 (B) and MT1-MMP (C) in the wt and crt−/− cells. Cells were cultured in conditioned media containing wortmannin (Wort.) and insulin (Ins) followed by lysis and RNA isolation using Trizol™ reagent. RT-PCR was carried out using primers specific to murine MMP-2 (A), MMP-9 (B), MT1-MMP (C) and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9, MT1-MMP (C) and GAPDH were quantified by densitometry using QuantityOne Program. Bar graph shows the mean ± SE of the MMP/GAPDH signal from 4 independent experiments. (D) A representative zymograph showing changes in the activity of MMP-2 and MMP-9 in conditioned media from the wt and crt−/− cells treated with DMEM + 0.2% DMSO, DMEM + 0.2% DMSO + insulin, and wortmannin. †P b 0.05 significantly different compared to the wt. ⁎P b 0.001 significantly different compared to the corresponding untreated cells.
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Fig. 8. Effect of MEK inhibitor on MMP-2 and MMP-9 expression in the wt and crt−/− cells. Cells were cultured in conditioned media containing MEK inhibitor (PD98059, PD) and insulin (Ins). Cells were lysed and RNA was isolated using Trizol™ reagent. Conditioned media were precipitated using trichloroacetic acid as described in Experimental procedures. RT-PCR was carried out using primers specific to murine MMP-2, MMP-9 and GAPDH as listed in Table 1. Bands corresponding to MMP-2, MMP-9 and GAPDH were quantified by densitometry using the QuantityOne Program. (A, C) Bar graphs showing the mean ± SE of the MMP/GAPDH signal from 4 different experiments. (B, D) Representative western blot showing changes in the MMP-2 (B) and MMP-9 (D) protein level in the conditioned media following treatment with PD98059. †P b 0.05 significantly different compared to the corresponding wt cells. ⁎P b 0.05 significantly different compared to the corresponding untreated cells.
MMP-2 and MMP-9 activities in both the wt and crt−/− cells as compared to the cells treated with conditioned media containing insulin (with 0.2% DMSO). Finally, the changes in the MMP activities correspond to the MMP-2 and MMP-9 mRNA expression in the wt and crt−/− cells (Fig. 7A, B, D). Inhibition of MEK (using PD98059) caused no change in the MMP-2 mRNA level in the wt (Fig. 8A) while crt−/− cells showed an increase in MMP-2 mRNA although not statistically significant. Fig. 8B shows that the level of MMP-2 protein secreted in the conditioned media also did not change significantly following PD98059 treatment. Intriguingly, inhibition of the MEK pathway by PD98059 and insulin resulted in a significant increase in the MMP-9 mRNA of the crt−/− cells with no change in the MMP-9 mRNA in the wt cells (Fig. 8C). The changes in the level of MMP-9 protein level in the conditioned media following PD98059 reflected that of MMP-9 mRNA (Fig. 8D). These data suggest that in the crt−/− cells insulin mediated activation of the Ras/Raf/MEK pathway inhibits the expression of MMP-9. At the same time, insulin's effect on activation of PI3 kinase elicits a significant increase in MMP-2 expression in the crt−/− cells. 3. Discussion Targeted deletion of the CRT gene has been shown to result in the thinning of the ventricular wall, increased intertrabecular recesses and the presence of omphaloceal in mouse embryos
(Mesaeli et al., 1999; Rauch et al., 2000). The failure of resorption of umbilical hernia (omphaloceal) implies that there are defects in wound healing process in the absence of CRT. Rauch et al. (2000) reported that ES derived crt−/− neuronal cells had defects in the wound healing process. Collectively, these defects could be due to altered extracellular matrix composition in the absence of CRT. However, to date no data are available on the effect of CRT in altering ECM composition or its degradation. MMP-2 and MMP-9 are two gelatinases which have been shown to regulate cardiac and vascular remodelling (Lijnen, 2001) and they have also been implicated in the regulation of wound healing (Simeon et al., 1999). In this study we showed, for the first time, a differential expression and activity of MMP-2 and MMP-9 in CRT deficient cells ((Figs. 1, 2, 4)). We have also demonstrated that partial restoration of CRT expression in the crt−/− cells could reverse the activities of MMP-2 and MMP-9 (Fig. 1B). Our data illustrate that these changes were not due to altered expression of endogenous inhibitors of MMP-2 and MMP-9 (TIMP-1 and TIMP-2, Fig. 5), but were due in part to altered PI3 kinase (Fig. 7) and/or MEK kinase activity (Fig. 8). MMP-2 and MMP-9 are both gelatinases which share a large number of extracellular matrix substrates (Nagase and Woessner, 1999), thus one might expect that the increase in MMP-2 activity and expression could compensate for the loss of MMP-9 activity in CRT deficient embryos and cells. However, our data show that in CRT deficient cells the elevated activity of MMP-2 is
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significantly lower than the attenuation of MMP-9 activity (Fig. 1). Overall, the additive effect of these changes will result in a significant loss of gelatinolytic activity in the crt−/− mice therefore it could contribute to the observed defects in the heart and omphaloceal formation in the crt−/− embryos (Mesaeli et al., 1999; Rauch et al., 2000). CRT is a molecular chaperone regulating the proper folding of many proteins in the ER (Ellgaard and Frickel, 2003; Guo et al., 2003). CRT is also involved in the regulation of intracellular Ca2+ homeostasis (Mery et al., 1996; Michalak et al., 1999) thus affecting expression of many genes at the transcriptional level (Waser et al., 1997). The decreased activity of MMP-9 in the absence of CRT (Fig. 1) therefore suggests a defect in MMP-9 protein synthesis or folding. However, we showed that these changes reflect a decrease in MMP-9 expression at the level of both the protein (Figs. 2 and 3) and mRNA (Fig. 4) in the crt−/− cells. On the other hand, cellular content of MMP-2 protein was not different between the crt−/− and wt cells, which could be due to shedding of the activated MMP-2 from the cell surface as was reported previously (Strongin et al., 1995). Increased expression of MT1-MMP has also been shown to activate proteolysis of pro-MMP-2 and release of the active MMP-2 in the extracellular space (Hofmann et al., 2000; Zhang and Brodt, 2003). Therefore, our observation on increased activity of MMP-2 in the crt−/− cells could be due to a combination of both increased MMP-2 expression and MT1-MMP expression and activity (Figs. 4 and 5). Two endogenously expressed inhibitory proteins TIMP-2 and TIMP-1 are also important in the regulation of MMP-2 and MMP-9 activity, respectively (Lafleur et al., 2003; Nagase and Woessner, 1999). In this study, we showed no changes in the expression of these two proteins in the crt−/− cells as compared to the wt cells (Fig. 5B). Therefore, the decreased activity of MMP-9 and increased MMP-2 activity are not due to changes in their inhibitory proteins. Furthermore, our results on corresponding changes in the protein and mRNA level of MMP-2 and MMP-9 in the crt−/− cells (Fig. 4) suggest that the effect of CRT on MMPs is not via its chaperone property but rather via transcriptional regulation of these genes. Previous studies showed that insulin and IGF-1 up-regulate MMP-2 expression in different cell types (Yoon and Hurta, 2001; Zhang et al., 2004), and this effect was due to both PI3 kinase dependent and independent pathways (Shin et al., 2005; Ulku et al., 2003; Yoon and Hurta, 2001). IGF receptor stimulation has also been shown to increase MT1-MMP activation in the Lewis lung carcinoma cell line (Zhang and Brodt, 2003). However, the role of insulin or IGF in the regulation of MMP-9 expression is not clear. In addition, the effect of the PI3 kinase activation on MMP-9 expression seems to be dependent on cell type. Glial derived neurotropic peptide has been shown to increase MMP-9 expression via a PI3-kinase dependent mechanism in MIA PaCa-2 cells (Okada et al., 2003). Whereas, PI3 kinase activation via the PDGF receptor was shown to inhibit cytokine (TNFα and IL-1) mediated increase in MMP-9 expression in C6 glioma cell line (Esparza et al., 1999). Recently, we have observed a significant increase in insulin receptor density and activation of the PI3 kinase/Akt activity in
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the crt−/− cells following insulin stimulation (Jalali and Mesaeli, manuscript under revision). Therefore, these changes in insulin receptor activity could in part explain the increased MMP-2 expression. This effect of insulin receptor/PI3 kinase could be at the level of both regulating the MMP-2 mRNA expression and activation of MT1-MMP which activates MMP-2 enzyme. A previous report has shown similar changes in the MMP-2 and MT1-MMP following stimulation of IGF receptors (Zhang and Brodt, 2003). Indeed, we showed that inhibition of PI3 kinase by wortmannin resulted in a significant inhibition of MMP-2 and MMP-9 expression (Fig. 7A, B) and activity (Fig. 7D) in the wt and crt−/− cells. However, the decrease in the MMP-2 expression was still significantly higher in the wortmannin treated crt−/− cells compared to the wortmannin treated wt cells. A role for insulin mediated activation of Ras/Raf/MEK in the modulation of MMP-2 and MMP-9 activities has also been reported previously (Shin et al., 2005; Yoon and Hurta, 2001). Indeed, here we have reported a significant increase in MMP-9 mRNA (Fig. 8C) and protein (Fig. 8D) in the crt−/− cells following treatment with MEK inhibitor PD98059. Furthermore, this treatment had no significant effect on the mRNA level of MMP-2 or MMP-9 in the wt cells (Fig. 8). These data suggest involvement of Ras/Raf/MEK pathway in the regulation of MMP-9 gene in the absence of CRT. To date, no data are available on the effect of CRT on the regulation of Ras/Raf/MEK pathway. Our current report is the only study suggesting alteration in these pathways in the absence of CRT. In addition to these two pathways other regulatory pathways such as NFκB (Knipp et al., 2004; Ogawa et al., 2004), intracellular calcium (Lohi and KeskiOja, 1995; Mukhopadhyay et al., 2004), and nitric oxide (MarcetPalacios et al., 2003) have been shown to regulate the expression of MMP-2 and MMP-9. Further investigations are needed to examine the role of these pathways in the regulation of MMP-2 and MMP-9 expression upon loss of CRT function. In conclusion, we showed that targeted deletion of CRT gene results in up-regulation of MMP-2 and MT1-MMP expression and activity accompanied by decreased MMP-9 expression and activity. These changes were partially due to activation of PI3 kinase dependent pathways and alteration of the Ras/Raf/MEK activities. 4. Experimental procedures 4.1. Materials Wortmannin, SB203580 and PD98059 were purchased from Calbiochem (EMD Biosciences Inc., USA). MMP-9 and actin antibodies were purchased from SigmaAldrich (St. Louis, USA). MMP-2 antibody was from MEDICORP (Montreal, QB, Canada), and DAB and ABC kit was from Vector Laboratories (Burlington, ON, Canada). Cell culture media and insulin–transferrin were from Invitrogen (Canada). 4.2. Cell culture and treatment Mouse embryonic fibroblast cells (MEF) from wild type (wt) and calreticulin null (crt−/−) 14-day-old mouse embryos were
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prepared as described previously (Mesaeli and Phillipson, 2004; Mesaeli et al., 1999). For experiments described below, cells were seeded at a density of 2 × 106 cells per plate and cultured in DMEM with 10% FBS at 37 °C. The next day, the cells were washed with PBS and incubated in conditioned media containing DMEM and 1% insulin–transferrin–selenium-A (Invitrogen) for 48 h. To examine the effect of reversal of CRT expression on MMP activities, crt−/− cells were transiently transfected with 10 μg of a plasmid encoding CRT-HA [described in Mesaeli and Phillipson, 2004] or an empty parental plasmid using Lipofectamine 2000. After 24 h the cells were washed and conditioned media were added and zymography experiments were carried out as described below. To determine the effect of different kinase pathways on the MMP-2 and MMP-9 mRNA expression, cells were cultured in conditioned media (DMEM containing 1% insulin–transferrin– selenium-A) for 24 h. The cells were washed with insulin free media and pre-incubated in the presence of specific inhibitors (10 nM PI3 kinase inhibitor wortmannin, or 50 μM MEK inhibitor PD98059 dissolved in DMSO) in insulin free media for 30 min. Next, the media was changed to DMEM containing the specific inhibitor and insulin–transferrin–selenium-A. In some experiments 0.2% DMSO was added to DMEM to examine the effect of the DMSO vehicle on MMP-2 and MMP-9 activities. The treated cells were then used to isolate RNA as described below. The conditioned media from these cells were used for examining the level of MMP-2 and MMP-9 as described below. 4.3. Zymography Conditioned media from the treated cells, non-treated cells and plates containing no cells (media control) were harvested and centrifuged at 2000 rpm for 10 min at room temperature. The collected media were then used for zymography. Twenty microliters of conditioned media was mixed with sample buffer containing 20% glycerol, 4% SDS, 0.13 M Tris, pH 6.8 and resolved on a 7.5% polyacrylamide gel containing 1 mg/ml porcine gelatin (Sigma). After electrophoresis, the gels were washed twice with 2.5% TritonX-100 for 30 min at room temperature to remove the SDS, followed by incubation in a buffer containing; 50 mM Tris–HCl, 5 mM CaCl2, 0.2 M NaCl, pH 7.6, at 37 °C for 48 h. The gels were then stained in Coomassie blue R-250 for 30 min at room temperature and destained with 40% methanol and 10% acetic acid over night at room temperature. Clear zones against the blue background indicated gelatinase activity. Purified human MMP-2 and MMP-9 (Chemicon International) were used as markers for zymography gels. To quantify the gelatinase activity, the stained zymography gels were scanned and the density of clear bands was calculated using the BioRad QuantityOne program. 4.4. Immunohistochemistry Paraffin sections (4 μm) from wt and crt−/− mouse embryos were used for immunohistochemistry using the ABC Kit (Vector Laboratories Inc.). Embryo sections were incubated
with rabbit anti-MMP-2 (1:200), or rabbit anti-MMP-9 (1:300) for 1 h at room temperature, followed by incubation with biotinylated anti-rabbit secondary antibody for 1 h at room temperature. The sections were stained with DAB (diaminobenzidine, Sigma) and nuclei were counter stained with haematoxylin QS (Vector Laboratories Inc.). Images were taken using a Zeiss Axioscop microscope using Axiovision software. 4.5. Western blot Cell were lysed using RIPA buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 0.5% NaDOC, 0.1% SDS, 0.15 M NaCl, pH 7.5) containing phenylmethysulfonyl fluoride (PMSF) and a cocktail of protease inhibitors (Sigma) (Mesaeli et al., 1999). Subsequently, the cell lysates were centrifugated at 7000 rpm, 4 °C for 5 min. The protein concentration of the supernatant was determined by a BioRad protein assay kit, using BSA as a standard. 10–50 μg protein was separated on 7.5% SDS– acrylamide gel. Western blot was performed using an antibody to MMP-2 or MMP-9 (Sigma) and Supersignal Chemiluminescent Substrate. Bands corresponding to MMP-2 and MMP-9 were quantified using QuantityOne program (BioRad). The level of MMP-2 and MMP-9 in conditioned media was analyzed following trichloroacetic acid precipitation. Briefly, after treatment of the cells with different inhibitors the total volume of the conditioned media mixed with 7.5% (v/v) trichloroacetic acid and 0.02% (v/v) sodium deoxycholate solutions. The mixture was incubated at 4 °C for 1 h followed by centrifugation at 13,200 rpm for 10 min at 4 °C. Protein pellets were washed several times with ice cold acetone, dried and re-suspended in Laemmli buffer. The proteins were then resolved on 7.5% SDS–acrylamide gel followed by western blot with MMP-2 and MMP-9 antibodies. 4.6. Reverse transcriptase assay Total RNA was isolated from the wt and crt−/− cells using Trizol™ reagent (Invitrogen). SuperScript™ II RT and Poly-dT oligo were used to generate cDNA from the RNA. MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2 and GAPDH were amplified using the Platinum Taq DNA polymerase (Invitrogen)
Table 1 List of primers used for RT-PCR Primer MMP-2 MMP-9 MT1-MMP TIMP-1 TIMP-2 GAPDH
Sequence Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense
5′-CATCGCCCATCATCAAGTTCC-3′ 5′-CCGAGCAAAAGCATCATCCAC-3′ 5′-TGATGCCGGCAGACAATCC-3′ 5′-CCTTATCCACGCGAATGACG-3′ 5′-ATGAAGAATTCAGGGCAGTGG-3′ 5′-GACCTTGTCCAGCAGCGAACGC-3′ 5′-TAAGGCCTGTAGCTGTGCC-3′ 5′-TCTTCATGCGGTTCTGGG-3′ 5′-TTTGCAATGCAGACGTAGTG-3′ 5′-TTCCTCCAACGTCCAGCG-3′ 5′-GCCAAGGTCATCCATGACAAC-3′ 5′-GTCCACCACCCTGTTGCTGTA-3′
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and gene specific primers listed in Table 1. In preliminary experiments the conditions of RT-PCR were optimized to determine the cycle number and annealing temperatures required for each primer set to obtain best results. Equal aliquots of the PCR reaction were resolved on a 1.5% agarose gel and bands corresponding to each gene listed above and GAPDH of the same sample were quantified using the BioRad QuantityOne Program. The data were then presented as the ratio of signal normalized to GAPDH signal (mean ± SE). Data were analyzed using the Student's t-test. Acknowledgment We thank Mr. Michael Wiwchar for superb technical assistance. This work was supported by a grant to N.M. from the Canadian Institute of Health Research (CIHR MOP# 42424). M.W. was the recipient of a fellowship from the University of Manitoba, H.M. is a Postdoctoral fellow of Canadian Institute of Health Research. N.M. is a new Investigator of Heart and Stroke Foundation of Canada. References Bornstein, P., Agah, A., Kyriakides, T.R., 2004. The role of thrombospondins 1 and 2 in the regulation of cell–matrix interactions, collagen fibril formation, and the response to injury. Int. J. Biochem. Cell Biol. 36, 1115–1125. Cowell, S., Knauper, V., Stewart, M.L., D'Ortho, M.P., Stanton, H., Hembry, R.M., Lopez-Otin, C., Reynolds, J.J., Murphy, G., 1998. Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3. Biochem. J. 331 (Pt 2), 453– 458. Creemers, E.E., Cleutjens, J.P., Smits, J.F., Daemen, M.J., 2001. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ. Res. 89, 201–210. Donnini, S., Morbidelli, L., Taraboletti, G., Ziche, M., 2004. ERK1–2 and p38 MAPK regulate MMP/TIMP balance and function in response to thrombospondin-1 fragments in the microvascular endothelium. Life Sci. 74, 2975–2985. Egeblad, M., Werb, Z., 2002. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev., Cancer 2, 161–174. Ellgaard, L., Frickel, E.M., 2003. Calnexin, calreticulin, and ERp57: teammates in glycoprotein folding. Cell Biochem. Biophys. 39, 223–247. Esparza, J., Vilardell, C., Calvo, J., Juan, M., Vives, J., Urbano-Marquez, A., Yague, J., Cid, M.C., 1999. Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways. Blood 94, 2754–2766. Guo, L., Groenendyk, J., Papp, S., Dabrowska, M., Knoblach, B., Kay, C., Parker, J.M., Opas, M., Michalak, M., 2003. Identification of an N-domain histidine essential for chaperone function in calreticulin. J. Biol. Chem. 278, 50645–50653. Hofmann, U.B., Westphal, J.R., Van Kraats, A.A., Ruiter, D.J., Van Muijen, G.N., 2000. Expression of integrin alpha(v)beta(3) correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo. Int. J. Cancer 87, 12–19. Knipp, B.S., Ailawadi, G., Ford, J.W., Peterson, D.A., Eagleton, M.J., Roelofs, K.J., Hannawa, K.K., Deogracias, M.P., Ji, B., Logsdon, C., Graziano, K.D., Simeone, D.M., Thompson, R.W., Henke, P.K., Stanley, J.C., Upchurch Jr., G.R., 2004. Increased MMP-9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-kappaB and activator protein-1 activity. J. Surg. Res. 116, 70–80.
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