Effect of exogenous heparin on the expression of heparanase
Heparin upregulated heparanase mRNA expression. Figure 1A shows an increase of 250-fold on heparanase mRNA expression with 500 µg/mL of heparin compared to 100 µg/mL. Moreover, heparin compared to 100 µg/mL treatment produced an increase of 4 times over the control.
Figure 1B shows that exogenous heparin also upregulates heparanase activity in the CHO-K1 cell line. The enzymatic activity of heparanase was enhanced 2 times with 500 µg/mL of heparin over 100 µg/mL of heparin, while an increase of 1.5 was observed between 100 µg/mL of heparin and the control (Fig. 1B). It is important to point out that heparin is a competitive inhibitor of heparanase. However, after heparin treatment, the culture medium was removed, and the cells harvested to determine the enzymatic activity using biotinylated heparan sulfate. Therefore, a competitive heparin inhibition scenario was set aside in the present assay.
These results suggest that the effect of heparin on mRNA expression and heparanase protein levels is concentration dependent.
Piva and co-workers showed that the depletion of glycosaminoglycan biosynthesis using 4-MU promoted a decrease of heparanase levels, indicating that glycosaminoglycans modulate heparanase since 4-MU inhibits glycosaminoglycans synthesis (19).
Mechanisms involved in heparanase modulation by heparin
The Wnt pathway and beta-catenin play a crucial role in several cellular processes, such as survival, migration, proliferation, and differentiation. It is conceivable that heparan sulfate proteoglycans act as co-receptors for a variety of ligands that regulate cell signaling. It is well known that heparan sulfate has high-affinity binding to Wnt (38). Furthermore, exogenous heparanase has been shown to modulate cellular responses to Wnt3a (39).
The Wnt/beta-catenin signaling controls the transcription of genes through the binding of a complex of beta-catenin and TCF to specific promoter elements. The activity of this final step in the Wnt/beta-catenin cascade can be investigated using a luciferase reporter construct. Thus, CHO-K1 cells were transiently transfected with the pTOPFLASH or pFOPFLASH reporter (30).
Heparin increased beta-catenin/TCF signaling as shown in Fig. 2. There was a 1.5-fold increase in luciferase activity after incubation with different concentrations of heparin compared to control (Fig. 2). The stimulation of Wnt activity by heparin was similar to LiCl treatment. An increase in Wnt activity corroborates with a decreased phosphorylation of beta-catenin as shown in Fig. 3.
To confirm that the Wnt/beta-catenin pathway is involved in the activation of heparanase expression, CHO-K1 cells were treated with LiCl, as shown in Fig. 4.
In addition, zebrafish embryos were treated with LiCl, the heparanase expression increased comparing to control group (Fig. 5).
Even though it is recognized that LiCl can inhibit other targets, its major effect has long been demonstrated to be GSK-3 inhibition in diverse organisms and in vitro (36). In addition, LiCl can modulate GSK-3/ phosphorylation on Ser9/21 by a complex mechanism. In this respect, studies have shown that inhibition of GSK-3 by lithium leads to increased N-terminal phosphorylation of GSK-3 and GSK-beta, demonstrating autoregulation of GSK-3 N-terminal serine phosphorylation. Thus, GSK-3 autoregulation could involve inhibition of a kinase or activation of a phosphatase. In the response to lithium chloride, this autoregulation implies two levels of inhibition: rapid response, direct inhibition of GSK-3 by lithium, followed by inactivation of the protein phosphatase (PP1). As a consequence of PP1 inactivation there is an increase in inhibitory phosphorylation of GSK-3 (37). Moreover, GSK-3 function in the Wnt pathway appears to be insulated from the effects of inhibitory N-terminal kinases such as Akt. It has been suggested that this inhibitory phosphorylation of GSK-3 would primarily affect Wnt-independent GSK3-regulated pathways, indicating an unexpected level of signaling pathway selectivity to lithium response.
There was a shift in the curves of heparanase fluorescence as shown in Flow Cytometer analysis, median 213 (control), 273 (treatment with heparin), 385 (treatment with LiCl), suggesting an increase in heparanase protein expression compared to CHO-K1 cells without any treatment (Control), as shown in Fig. 4.
In addition, after treatment with LiCl, zebrafish embryos presented an enhance of heparanase expression, confirming that the same mechanism occurs in vivo, as demonstrated in Fig. 5.
The mechanisms that modulate heparanase activity and heparanase expression are still unclear. It is important to emphasize that heparin is a known inhibitor of heparanase activity since it competes with heparan sulfate for the active site of the enzyme (23). Nevertheless, in the present study the treatment of CHO-K1 cells with heparin induced an increase in mRNA expression and protein level.