Heparanase regulates levels of syndecan-1 in the nucleus - PubMed (original) (raw)

Heparanase regulates levels of syndecan-1 in the nucleus

Ligong Chen et al. PLoS One. 2009.

Abstract

Syndecan-1 is a transmembrane heparan sulfate-bearing proteoglycan known to regulate multiple biological functions at the cell surface and within the extracellular matrix. Its functional activity can be modulated by heparanase, an enzyme that cleaves heparan sulfate chains and whose expression has been associated with an aggressive phenotype in many cancers. In addition to remodeling syndecan-1 by cleaving its heparan sulfate chains, heparanase influences syndecan-1 location by upregulating expression of enzymes that accelerate its shedding from the cell surface. In the present study we discovered that heparanase also alters the level of nuclear syndecan-1. Upon upregulation of heparanase expression or following addition of recombinant heparanase to myeloma cells, the nuclear localization of syndecan-1 drops dramatically as revealed by confocal microscopy, western blotting and quantification by ELISA. This effect requires enzymatically active heparanase because cells expressing high levels of mutated, enzymatically inactive heparanase, failed to diminish syndecan-1 levels in the nucleus. Although heparan sulfate function within the nucleus is not well understood, there is emerging evidence that it may act to repress transcriptional activity. The resulting changes in gene expression facilitated by the loss of nuclear syndecan-1 could explain how heparanase enhances expression of MMP-9, VEGF, tissue factor and perhaps other effectors that condition the tumor microenvironment to promote an aggressive cancer phenotype.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Syndecan-1 is not detected within the nucleus of cells expressing high levels of heparanase.

Confocal microscopic z-stack images of (A) HPSE-low and (B) HPSE-high cells immunostained using antibody to syndecan-1. Blue (Hoechst stain) identifies nuclei; white identifies syndecan-1 within the nucleus (co-localization of Hoechst and syndecan-1); green identifies cytoplasmic and cell surface syndecan-1. Syndecan-1 is detected within nuclei of HPSE-low cells but absent in nuclei of the HPSE-high cells. Bar = 10 µm. Note: As is characteristic of myeloma cells, the size of the nucleus is large relative to the amount of cytoplasm.

Figure 2

Figure 2. Elevated expression of heparanase dramatically decreases the level of syndecan-1 present within the nucleus.

A) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pcDNA3 vector for transfections) and separated on SDS-PAGE. Western blots were probed with antibody to human syndecan-1, human heparanase, SP-1 or actin. B) Nuclear and non-nuclear fractions were isolated from HPSE-low and HPSE-high cells (prepared using the pIRES2 vector for transfections) and from wild-type CAG cells infected with control shRNA or an shRNA targeting heparanase. The quantity of syndecan-1 in each fraction was determined by ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P<0.01 vs. nuclear syndecan-1 in HPSE low cells; **, P<0.02 vs. nuclear syndecan-1 in shRNA control.

Figure 3

Figure 3. Heparanase enzymatic activity is required for reduction of syndecan-1 levels in the nucleus.

Nuclear and non-nuclear fractions were prepared from CAG cells expressing high levels of wild-type heparanase (HPSE-high) or heparanase mutated at either amino acid 343 (M343) or amino acid 225 (M225) which renders them enzymatically inactive. All cells were prepared using pIRES2 vectors for transfections. Fractions were analyzed for syndecan-1 levels by A) western blotting and B) ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P<0.01 vs. nuclear syndecan-1 in HPSE high cells.

Figure 4

Figure 4. Exogenous recombinant heparanase (rHPSE) decreases nuclear syndecan-1 levels in a concentration-dependent manner.

Recombinant heparanase was added to CAG cells having very low levels of heparanase expression (shRNA knockdown cells). Nuclear and non-nuclear fractions were prepared and syndecan-1 levels analyzed by A) western blotting and B) ELISA. Grey bars = non-nuclear fraction; Black bars = nuclear fraction. Error bars represent standard error of the mean. *, P<0.01 vs. nuclear syndecan-1 in cells treated with 0 ng/ml rHPSE.

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