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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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Expression of MMPs in heparanase transfected MDA-231 human breast carcinoma cells.MDA-MB-231 cells were transfected with a mock (empty vector) or either active (Hpse) or mutated inactive (Mut) heparanase gene. Heparanase (A) and MMPs (B) mRNA expression levels were determined by real-time PCR, as described under “Materials and Methods”. The appropriate primers are listed in Table 2. The expression levels determined in the mock transfected cells were regarded as 100%, and the levels in Hpse and mut-Hpse transfected cells were presented as percentage relative to the mock transfected cells. Decreased levels of MMP-2, MMP-9, and MMP-14 mRNAs were noted in cells over-expressing the active form of heparanase, but not the double mutant, inactive form of the enzyme.
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pone-0005181-g008: Expression of MMPs in heparanase transfected MDA-231 human breast carcinoma cells.MDA-MB-231 cells were transfected with a mock (empty vector) or either active (Hpse) or mutated inactive (Mut) heparanase gene. Heparanase (A) and MMPs (B) mRNA expression levels were determined by real-time PCR, as described under “Materials and Methods”. The appropriate primers are listed in Table 2. The expression levels determined in the mock transfected cells were regarded as 100%, and the levels in Hpse and mut-Hpse transfected cells were presented as percentage relative to the mock transfected cells. Decreased levels of MMP-2, MMP-9, and MMP-14 mRNAs were noted in cells over-expressing the active form of heparanase, but not the double mutant, inactive form of the enzyme.

Mentions: To further investigate the interrelation between heparanase and MMPs, we transfected human breast carcinoma MDA-NB-231 cells, normally expressing moderate levels of heparanase [22], with either active or double mutant heparanase (active site Gln 225 and Gln 343 replaced by Ala) lacking enzymatic activity [41]. Both, the active and inactive mutant heparanase were ∼30 fold over-expressed in the Hpse-transfected cells as compared to mock transfected cells (Fig. 8A). As demonstrated in Fig. 8B, cells transfected with active heparanase exhibited a marked decrease in expression of MMP-2 (5.8 fold), MMP-9 (6.5 fold) and MMP-14 (3 fold), a mirror image of the increased expression found in Hpse-KO mice. In contrast, transfecting the MDA-231 cells with the double mutant inactive heparanase did not affect MMP expression (Fig. 8B), indicating that heparanase enzymatic activity is involved in the observed regulation of MMP expression.


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)

Expression of MMPs in heparanase transfected MDA-231 human breast carcinoma cells.MDA-MB-231 cells were transfected with a mock (empty vector) or either active (Hpse) or mutated inactive (Mut) heparanase gene. Heparanase (A) and MMPs (B) mRNA expression levels were determined by real-time PCR, as described under “Materials and Methods”. The appropriate primers are listed in Table 2. The expression levels determined in the mock transfected cells were regarded as 100%, and the levels in Hpse and mut-Hpse transfected cells were presented as percentage relative to the mock transfected cells. Decreased levels of MMP-2, MMP-9, and MMP-14 mRNAs were noted in cells over-expressing the active form of heparanase, but not the double mutant, inactive form of the enzyme.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664924&req=5

pone-0005181-g008: Expression of MMPs in heparanase transfected MDA-231 human breast carcinoma cells.MDA-MB-231 cells were transfected with a mock (empty vector) or either active (Hpse) or mutated inactive (Mut) heparanase gene. Heparanase (A) and MMPs (B) mRNA expression levels were determined by real-time PCR, as described under “Materials and Methods”. The appropriate primers are listed in Table 2. The expression levels determined in the mock transfected cells were regarded as 100%, and the levels in Hpse and mut-Hpse transfected cells were presented as percentage relative to the mock transfected cells. Decreased levels of MMP-2, MMP-9, and MMP-14 mRNAs were noted in cells over-expressing the active form of heparanase, but not the double mutant, inactive form of the enzyme.
Mentions: To further investigate the interrelation between heparanase and MMPs, we transfected human breast carcinoma MDA-NB-231 cells, normally expressing moderate levels of heparanase [22], with either active or double mutant heparanase (active site Gln 225 and Gln 343 replaced by Ala) lacking enzymatic activity [41]. Both, the active and inactive mutant heparanase were ∼30 fold over-expressed in the Hpse-transfected cells as compared to mock transfected cells (Fig. 8A). As demonstrated in Fig. 8B, cells transfected with active heparanase exhibited a marked decrease in expression of MMP-2 (5.8 fold), MMP-9 (6.5 fold) and MMP-14 (3 fold), a mirror image of the increased expression found in Hpse-KO mice. In contrast, transfecting the MDA-231 cells with the double mutant inactive heparanase did not affect MMP expression (Fig. 8B), indicating that heparanase enzymatic activity is involved in the observed regulation of MMP expression.

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