PH20 Inhibits TGFβ1-Induced Differentiation of Perimysial Orbital Fibroblasts via Hyaluronan-CD44 Pathway in Thyroid-Associated Ophthalmopathy.

Abstract

Hyaluronan (HA) is a potential regulator of TGFβ1-induced differentiation in perimysial orbital fibroblasts (pOFs). Our study aimed to explore the effects of PH20 (a hyaluronidase) and HA on TGFβ1-induced differentiation in pOFs cultured from thyroid-associated ophthalmopathy (TAO) and control subjects. TAO and control pOFs (passages 3-6) were incubated with TGFβ1 ± PH20 or TGFβ1 ± HA for 72 hours and processed for various analyses, including HA electrophoresis; αSMA immunofluorescence; and quantification of αSMA (encoded by ACTA2), CD44 (a cell surface HA receptor), and SMAD2/3 (signaling molecule in the TGFβ1 pathway). TGFβ1 induced myogenic differentiation (marked by αSMA upregulation) of pOFs. After TGFβ1 treatment, more HA accumulated in the TAO group than in the control group. PH20 mainly digested medium to small HA and increased the percentage of high molecular weight HA (HMW-HA) in total HA. Both PH20 and HMW-HA inhibited TGFβ1-induced differentiation in the TAO group, but neither showed significant effects in the control group. CD44 level negatively correlated with ACTA2 level in the TAO group, but no correlation was detected in the control group. Both PH20 and HMW-HA upregulated CD44 expression and inhibited SMAD2/3 expression in the TAO group, and the inhibitory effects were partially reversed by CD44 blockage. CD44 level in the control group was not affected by PH20 or HMW-HA treatment, and CD44 blockage showed no significant effects on control pOF differentiation. PH20 inhibits TGFβ1-induced differentiation of TAO pOFs via HA-CD44-SMAD2/3 signaling, and the HA-CD44 signaling plays a divergent role in TAO and control pOFs.