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. 2010 Dec 1;111(5):1299-309.
doi: 10.1002/jcb.22854.

Modulation of GEF-H1 induced signaling by heparanase in brain metastatic melanoma cells

Lon D Ridgway  1 , Michael D Wetzel, Dario Marchetti
Affiliations

Modulation of GEF-H1 induced signaling by heparanase in brain metastatic melanoma cells

Lon D Ridgway et al. J Cell Biochem. .

Abstract

Mechanisms of brain metastatic melanoma (BMM) remain largely unknown. Understanding the modulation of signaling pathways that alter BMM cell invasion and metastasis is critical to develop new therapies for BMM. Heparanase has been widely implicated in cancer and is the dominant mammalian endoglycosidase which degrades heparan sulfate chains of proteoglycans (HSPG) including syndecans (SDCs). Recent findings also indicate that heparanase possesses non-enzymatic functions in its latent form. We hypothesized that extracellular heparanase modulates BMM cell signaling by involving SDC1/4 carboxy terminal-associated proteins and downstream targets. We digested BMM cell surface HS with human recombinant active or latent heparanase to delineate their effects on cytoskeletal dynamics and cell invasiveness. We identified the small GTPase guanine nucleotide exchange factor-H1 (GEF-H1) as a new component of a SDC signaling complex that is differentially expressed in BMM cells compared to corresponding non-metastatic counterparts. Second, knockdown of GEF-H1, SDC1, or SDC4 decreased BMM cell invasiveness and GEF-H1 modulated small GTPase activity of Rac1 and RhoA in conjunction with heparanase treatment. Third, both active and latent forms of heparanase affected Rac1 and RhoA activity; notably increasing RhoA activity. Both forms of heparanase were found to mediate the expression and subcellular localization of GEF-H1, and treatment of BMM with latent heparanase modulated SDC1/4 gene expression. Finally, treatment with exogenous heparanase downregulated BMM cell invasion. These studies indicate the relevance of heparanase signaling pathways in BMM progression, and provide insights into the molecular mechanisms regulating HSPG signaling in response to exogenous heparanase.

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Conflict of interest statement

The authors declare no competing conflicts of interest.

Figures

Fig. 1
Fig. 1
Syndecan profiling in melanoma cells. A: Total RNA was extracted from brain metastatic SB1B or syngeneic non-metastatic SBCl3 melanoma cells, and levels of the four mammalian syndecans (SDCs) were determined by RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a positive control. B: Protein expression of SDC1/4 in BMM cells. Whole cell lysates treated with heparitinase and chondroitinase from SBCl3 and SB1B cells were Western blotted for SDC1/4 to determine syndecan core protein levels. β-Actin was used as a loading control. See Materials and Methods Section for additional details.
Fig. 2
Fig. 2
Differential expression of SDC by exogenous heparanase. A: Heparanase activity plot. Concentration of active HPSE used in experiments is indicated by the red box. Heparanase was prepared as described in the Materials and Methods Section. B: Heparanase regulates SDC gene expression. RT-PCR was performed on RNA from BMM cells to determine changes in SDC gene expression level. Cells were treated with or without 500 ng/ml of active or latent heparanase (A-HPSE or L-HPSE, respectively) for 18 h at 37°C, then RNA was isolated, followed by semi-quantitative RT-PCR. Densitometric values represent expression of SDC1/4 normalized to GAPDH. C: Effects of HPSE on SDC expression. Cells were treated with or without active or latent (A- or L-HPSE) (500 ng/ml) for 16 h at 37°C, then whole cell lysates were treated with hepartinase and chondroitinase for 4 h at 37°C, then Western blotted with antibodies against SDC1/4, heparanase, and β-actin (loading control). See Materials and Methods Section for additional details.
Fig. 3
Fig. 3
GEF-H1 as a SDC-associated proteins. A: Expression levels of GEF-H1 and PKCα in SBCl3 and SB1B BMM cells. Whole cell lysates from BMM cells treated with A-HPSE or L-HPSE were Western blotted for GEF-H1 or PKCα to determine protein expression levels in different BMM cell lines. β-Actin was used as a loading control. Densitometric analyses represent GEF-H1 or PKCα levels normalized to β-actin. *P-value <0.05, **P-value <0.01, comparing SBCl3 to SB1B expression levels. B: GST pulldown of GEF-H1 by SDC1 and SDC4. BMM cells were treated with or without 500 ng/ml of A-HPSE or L-HPSE for 18 h at 37°C, then whole cell lysates were generated, passed over GST-SDC1/4 CT fusion protein affinity columns. This was followed by immunoblotting for GEF-H1. See Materials and Methods Section for additional details.
Fig. 4
Fig. 4
Effects of GEF-H1 and SDC1/4 on BMM cell invasion. A: Knockdown of GEF-H1 in BMM cells. SBCl3 cells were treated with SmartPool siRNA (Dharmacon) for GEF-H1 or scrambled control, and protein level was determined by Western blotting. Knockdown of SDC1 (B) and SDC4 (C) in BMM cells. BMM cells (SBCl3 and SB1B) were treated with siRNA for SDC1 or scrambled control and Western blotted to determine SDC1 knockdown. β-Actin was used as a loading control for all samples. C: Effects of inhibition of GEF-H1 and SDCs on BMM cell invasion. Cells were treated with siRNA for GEF-H1, SDC1, or SDC4, or scrambled control for 36 h, then plated on Matrigel™ coated inserts and allowed to invade for 24 h. Invasion was determined by crystal violet staining followed by extraction and OD readings were made at 490 nm. Data are representative of three independent experiments and presented as means ± standard deviations. *P-value <0.05, **P-value <0.01. Statistical analyses for each sample were made by comparing GEF-H1, SDC1, or SDC4 siRNA treated cells versus scrambled control for each cell line. See Materials and Methods Section for additional details.
Fig. 5
Fig. 5
Roles of GEF-H1, SDCs and Heparanase in BMM cell Rac1/RhoA activities. A: Expression of Rac1 and RhoA small GTPases in BMM cells. Differential activation of Rac1 (B) and RhoA (C) in BMM cells in response to 10% (v/v) serum-containing media (GM). Positive and negative controls of non-hydrolyzed GTP were from the Rac and Rho Activation Assays (Millipore) are indicated by + and −, respectively. The upper band represents the phosphorylated form of the protein. D: Effects of HPSE on Rac1 and RhoA. SBCl3 and SB1B cells were treated with or without 500 ng/ml A-HPSE or L-HPSE for 16 h at 37°C, then serum-starved for 24 h. Rac and Rho activities were determined using the Cytoskeleton G-LISA activation kits. E: Effects of HPSE on Rac1 and RhoA expression. SBCl3 and SB1B cells were treated with or without A-HPSE or L-HPSE, and whole cell lysates were analyzed for Rac1 and RhoA expression levels. F: Effects of GEF-H1 knockdown and heparanase on Rac1/RhoA activity. SBCl3 and SB1B cells were transfected with siRNA for GEF-H1 or scrambled control, then cells were treated with or without active or latent (A- or L-HPSE) (500 ng/ml) for 16 h at 37°C. Cells were then lysed and screened for Rac1/RhoA activity using the Cytoskeleton G-LISA activation kits. All data are represented as means ± standard deviations for three experiments. *P-value <0.05, **P-value <0.01. See Materials and Methods Section for additional details.
Fig. 6
Fig. 6
Roles of heparanase on GEF-H1 subcellular localization. Cells were treated with or without of A-HPSE or L-HPSE (500 ng/ml), then Western blotted for GEF-H1. Fibrillarin and GAPDH were used as specific nuclear and cytosolic markers respectively. See Materials and Methods Section for additional details.
Fig. 7
Fig. 7
Roles of heparanase in BMM cell invasiveness. SBCl3 and SB1B cells were treated with or without A-HPSE or L-HPSE (500 ng/ml) for 16 h at 37°C, then serum-starved for 24 h before being used for invasion assays. All data are represented as means ± standard deviations and are indicative of three independent experiments. *P-value <0.05, **P-value <0.01. See Materials and Methods Section for additional details.
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