Hyaluronan synthase in trabecular meshwork cells

Abstract

Background/aims: Hyaluronan is present in the trabecular meshwork where it is involved in the pathophysiology of aqueous outflow environment. In this study, the expression and regulation of hyaluronan synthase (HAS), which is the enzyme synthesising hyaluronan, in trabecular meshwork cells were investigated.

Methods: Cultured bovine trabecular meshwork cells (BTMCs) were used. HAS expression in BTMCs was examined by RT-PCR. The effects of transforming growth factor beta (TGF-beta) and platelet derived growth factor BB (PDGF-BB) on HAS expression in BTMCs were examined by quantitative RT-PCR. The HAS2 expression by TGF-beta and PDGF-BB at the protein level was also confirmed immunohistochemically. The production of hyaluronan from BTMCs was detected by high performance liquid chromatography (HPLC).

Results: Three HAS isoforms were expressed in BTMCs at the mRNA level. Among HAS isoforms, only the expression of HAS2 mRNA was increased by the administration of TGF-beta or PDGF-BB. HAS2 upregulation by these growth factors was also confirmed at the protein level. Further, hyaluronan production from BTMCs was stimulated by TGF-beta or PDGF-BB.

Conclusion: Expression of HAS in trabecular meshwork may maintain the hyaluronan content in the aqueous outflow pathway. Its production is regulated by TGF-beta and PDGF-BB. The regulation of the expression of HAS in trabecular meshwork might be useful for modulating the aqueous outflow environment.

Figure 1

Detection of HAS2 mRNA in response to external stimuli. HAS2 mRNA expression in response to 10 ng/ml of TGF-β1 or PDGF-BB in BTMCs was analysed by competitive PCR. Real time fluorescein PCR detection (LightCycler) was used in this experiment (n = 6, error bar is SD). The y-axis gives standardising log concentration values, which are obtained from a standard curve, and show the relative amount of HAS2 mRNA.

Figure 2

Immunohistochemical detection of HAS2 in BTMCs. Ethanol fixed BTMCs were incubated in the absence or presence of anti-HAS2 antibody and visualised by DAB chromogen. (A) Control IgG (isotype control), (B) no treatment, (C) treatment with 10 ng/ml of TGF-β1, (D) treatment with 10 ng/ml of PDGF-BB. BTMCs were weakly stained by anti-HAS2 polyclonal antibody in plasma membrane and partial cytoplasm without TGF-β stimulation (B). When BTMCs were incubated with growth factors, BTMCs strongly expressed the HAS2 isoform on the plasma membrane (C) and (D).

Figure 3

Cumulative signal strength in plasma membrane (A) and cytoplasm (B) was measured using Photoshop based densitometry.

Figure 4

TGF-β and PDGF-BB stimulates hyaluronan production in BTMCs. Quiescent cells were incubated for 24 hours in DMEM medium contained 1% FBS in the absence or presence of 10 ng/ml TGF-β1, TGF-β2, or PDGF-BB. Then, hyaluronan content in media was measured by high performance liquid chromatography (n=4, error bar is SD).