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Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix metalloproteinases.

Zcharia E, Jia J, Zhang X, Baraz L, Lindahl U, Peretz T, Vlodavsky I, Li JP - PLoS ONE (2009)

Bottom Line: Heparanase, a mammalian endo-beta-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix.Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells.It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Heparanase, a mammalian endo-beta-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix. This single gene encoded enzyme is over-expressed in most human cancers, promoting tumor metastasis and angiogenesis.

Principal findings: We report that targeted disruption of the murine heparanase gene eliminated heparanase enzymatic activity, resulting in accumulation of long heparan sulfate chains. Unexpectedly, the heparanase knockout (Hpse-KO) mice were fertile, exhibited a normal life span and did not show prominent pathological alterations. The lack of major abnormalities is attributed to a marked elevation in the expression of matrix metalloproteinases, for example, MMP2 and MMP14 in the Hpse-KO liver and kidney. Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells. Immunostaining (kidney tissue) and chromatin immunoprecipitation (ChIP) analysis (Hpse-KO mouse embryonic fibroblasts) suggest that the newly discovered co-regulation of heparanase and MMPs is mediated by stabilization and transcriptional activity of beta-catenin.

Conclusions/significance: The lack of heparanase expression and activity was accompanied by alterations in the expression level of MMP family members, primarily MMP-2 and MMP-14. It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate. Generation of viable Hpse-KO mice lacking significant abnormalities may provide a promising indication for the use of heparanase as a target for drug development.

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MMP expression in Hpse-KO mice.A. Real-time PCR. RNA was extracted from liver, kidney and mammary gland of wt and Hpse-KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, MMP-14 and MMP-25. The expression level determined for each MMP in the wt tissue (blue) was regarded as 100% and the corresponding expression determined in the Hpse-KO tissue (Purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Western blot analysis. Liver, kidney and mammary gland tissue extracts, prepared as described in “Materials and Methods”, were subjected to Western blot analysis using anti-mouse MMP-2 monoclonal antibodies (mA801B; upper panels), anti mouse β-catenin (mAb610154; middle panels), or anti mouse α-tubulin (B-5-1-2; lower panels). Higher protein levels of MMP-2 and β-catenin were detected in samples derived from Hpse-KO vs. wt tissues. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice. D. β-catenin immunostaining. Parafin embedded kidney tissue sections were subjected to immunostaining with antibody directed against β-catenin. Increased staining was observed in kidney derived from Hpse-KO vs. wt mice. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice.
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pone-0005181-g006: MMP expression in Hpse-KO mice.A. Real-time PCR. RNA was extracted from liver, kidney and mammary gland of wt and Hpse-KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, MMP-14 and MMP-25. The expression level determined for each MMP in the wt tissue (blue) was regarded as 100% and the corresponding expression determined in the Hpse-KO tissue (Purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Western blot analysis. Liver, kidney and mammary gland tissue extracts, prepared as described in “Materials and Methods”, were subjected to Western blot analysis using anti-mouse MMP-2 monoclonal antibodies (mA801B; upper panels), anti mouse β-catenin (mAb610154; middle panels), or anti mouse α-tubulin (B-5-1-2; lower panels). Higher protein levels of MMP-2 and β-catenin were detected in samples derived from Hpse-KO vs. wt tissues. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice. D. β-catenin immunostaining. Parafin embedded kidney tissue sections were subjected to immunostaining with antibody directed against β-catenin. Increased staining was observed in kidney derived from Hpse-KO vs. wt mice. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice.

Mentions: The unexpected result of abnormal mammary gland morphology and increased neovascularization in Hpse-KO mice led us to search for a possible explanation for this phenotype. The immediate question was whether other ECM degrading enzyme(s) were compensating for the lack of heparanase expression. Hpa2, a gene exhibiting significant homology (∼38%) to the heparanase gene [37], but lacking a detectable heparanase enzymatic activity, was the first candidate to examine. For this purpose, total RNA extracted from the kidney, liver and mammary gland of Hpse-KO and wt mice, was analyzed using specific primers corresponding to Hpa2 (Table 2). Analysis of Hpa2 expression in the different tissues revealed no significant difference between wt and Hpse-KO mice (Fig. 5). These results were further corroborated by Western blot of Hpa2 showing no difference in protein level between Hpse-KO and wt mice (not shown). Moreover, the increased HS length found in Hpse-KO mice does not support upregulation of additional heparanase-like enzyme. Taking into account that matrix metalloproteinases (MMPs) play important roles in rearranging the ECM structure and thereby in tissue remodeling, morphogenesis and neovascularization, we investigated the expression of MMPs (real-time PCR) in the Hpse-KO vs. wt mice. For this purpose, the RNA samples were further analyzed using specific primers corresponding to MMP-2, -3, -9, -14 and -25 (Table 2). The results (Fig. 6A) indicated that the lack of heparanase expression was associated with marked changes in the expression levels of several members of the MMP family. MMP-2 was over-expressed (2–3.5 fold) in all samples extracted from Hpse-KO vs. wt mice. MMP-14 was over-expressed (4–7 fold) in the liver and kidney, but down regulated (∼4 fold) in mammary glands derived from Hpse-KO vs. wt mice. MMP-9 and MMP-25 expression levels were altered, albeit to a lower extent, depending on the tissue (Fig. 6A).


Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix metalloproteinases.

Zcharia E, Jia J, Zhang X, Baraz L, Lindahl U, Peretz T, Vlodavsky I, Li JP - PLoS ONE (2009)

MMP expression in Hpse-KO mice.A. Real-time PCR. RNA was extracted from liver, kidney and mammary gland of wt and Hpse-KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, MMP-14 and MMP-25. The expression level determined for each MMP in the wt tissue (blue) was regarded as 100% and the corresponding expression determined in the Hpse-KO tissue (Purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Western blot analysis. Liver, kidney and mammary gland tissue extracts, prepared as described in “Materials and Methods”, were subjected to Western blot analysis using anti-mouse MMP-2 monoclonal antibodies (mA801B; upper panels), anti mouse β-catenin (mAb610154; middle panels), or anti mouse α-tubulin (B-5-1-2; lower panels). Higher protein levels of MMP-2 and β-catenin were detected in samples derived from Hpse-KO vs. wt tissues. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice. D. β-catenin immunostaining. Parafin embedded kidney tissue sections were subjected to immunostaining with antibody directed against β-catenin. Increased staining was observed in kidney derived from Hpse-KO vs. wt mice. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice.
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pone-0005181-g006: MMP expression in Hpse-KO mice.A. Real-time PCR. RNA was extracted from liver, kidney and mammary gland of wt and Hpse-KO mice and subjected to quantitative real time PCR analysis to evaluate the expression of MMP-2, MMP-9, MMP-14 and MMP-25. The expression level determined for each MMP in the wt tissue (blue) was regarded as 100% and the corresponding expression determined in the Hpse-KO tissue (Purple) are presented as percentage relative to it. Each reaction was repeated 6 times and the mean±SD is indicated. B. Western blot analysis. Liver, kidney and mammary gland tissue extracts, prepared as described in “Materials and Methods”, were subjected to Western blot analysis using anti-mouse MMP-2 monoclonal antibodies (mA801B; upper panels), anti mouse β-catenin (mAb610154; middle panels), or anti mouse α-tubulin (B-5-1-2; lower panels). Higher protein levels of MMP-2 and β-catenin were detected in samples derived from Hpse-KO vs. wt tissues. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice. D. β-catenin immunostaining. Parafin embedded kidney tissue sections were subjected to immunostaining with antibody directed against β-catenin. Increased staining was observed in kidney derived from Hpse-KO vs. wt mice. C. MMP2 zymography. Serum samples derived from Hpse-KO and wt mice were evaluated for MMP-2 activity. MMP-2 activity was approximately 3 fold higher in plasma samples derived from Hpse-KO vs. wt mice.
Mentions: The unexpected result of abnormal mammary gland morphology and increased neovascularization in Hpse-KO mice led us to search for a possible explanation for this phenotype. The immediate question was whether other ECM degrading enzyme(s) were compensating for the lack of heparanase expression. Hpa2, a gene exhibiting significant homology (∼38%) to the heparanase gene [37], but lacking a detectable heparanase enzymatic activity, was the first candidate to examine. For this purpose, total RNA extracted from the kidney, liver and mammary gland of Hpse-KO and wt mice, was analyzed using specific primers corresponding to Hpa2 (Table 2). Analysis of Hpa2 expression in the different tissues revealed no significant difference between wt and Hpse-KO mice (Fig. 5). These results were further corroborated by Western blot of Hpa2 showing no difference in protein level between Hpse-KO and wt mice (not shown). Moreover, the increased HS length found in Hpse-KO mice does not support upregulation of additional heparanase-like enzyme. Taking into account that matrix metalloproteinases (MMPs) play important roles in rearranging the ECM structure and thereby in tissue remodeling, morphogenesis and neovascularization, we investigated the expression of MMPs (real-time PCR) in the Hpse-KO vs. wt mice. For this purpose, the RNA samples were further analyzed using specific primers corresponding to MMP-2, -3, -9, -14 and -25 (Table 2). The results (Fig. 6A) indicated that the lack of heparanase expression was associated with marked changes in the expression levels of several members of the MMP family. MMP-2 was over-expressed (2–3.5 fold) in all samples extracted from Hpse-KO vs. wt mice. MMP-14 was over-expressed (4–7 fold) in the liver and kidney, but down regulated (∼4 fold) in mammary glands derived from Hpse-KO vs. wt mice. MMP-9 and MMP-25 expression levels were altered, albeit to a lower extent, depending on the tissue (Fig. 6A).

Bottom Line: Heparanase, a mammalian endo-beta-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix.Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells.It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Heparanase, a mammalian endo-beta-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix. This single gene encoded enzyme is over-expressed in most human cancers, promoting tumor metastasis and angiogenesis.

Principal findings: We report that targeted disruption of the murine heparanase gene eliminated heparanase enzymatic activity, resulting in accumulation of long heparan sulfate chains. Unexpectedly, the heparanase knockout (Hpse-KO) mice were fertile, exhibited a normal life span and did not show prominent pathological alterations. The lack of major abnormalities is attributed to a marked elevation in the expression of matrix metalloproteinases, for example, MMP2 and MMP14 in the Hpse-KO liver and kidney. Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells. Immunostaining (kidney tissue) and chromatin immunoprecipitation (ChIP) analysis (Hpse-KO mouse embryonic fibroblasts) suggest that the newly discovered co-regulation of heparanase and MMPs is mediated by stabilization and transcriptional activity of beta-catenin.

Conclusions/significance: The lack of heparanase expression and activity was accompanied by alterations in the expression level of MMP family members, primarily MMP-2 and MMP-14. It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate. Generation of viable Hpse-KO mice lacking significant abnormalities may provide a promising indication for the use of heparanase as a target for drug development.

Show MeSH
Related in: MedlinePlus