A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

Abstract

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in concentration dependent manner unlike the Dcn−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn−/− CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn−/− CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn−/− mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.

Chondroitin/dermatan 2-O sulfotransferase potentiates Fgf2 induced cell migration

“…We previously demonstrated that the reduced amount of Ust and the lack of 2-O sulfated CS/DS in skin or fibroblasts of Dcn 2/2 mice leads to an altered Fgf7-and Fgf2-binding capacity (Nikolovska et al, 2014), suggesting they have a new and important role in modulating the growth factor response. In the present study, we show that the degree of 2-O sulfation of cell surface CS/DS can modulate Fgf2-induced migration through ERK1/2 activation.…”

“…Recently, the small non-coding microRNA149 has been linked to a new fine-tuning element of Fgf2-induced proliferation in endothelial cells by regulating the HS proteoglycan glypican (Chamorro-Jorganes et al, 2014). We have previously shown that the lack of decorin in skin led to the reduction of Ust and an impaired cell morphology in a 3D environment (Nikolovska et al, 2014;Jungmann et al, 2012), which could partially contribute to the delayed wound healing of the Dcn 2/2 mice (Järveläinen et al, 2006). We proved the importance of 2-O sulfation by showing that there was an impaired migration of 3T3 cells with a knockdown of Ust.…”

“…To strengthen our hypothesis that a minor modification like CS/DS 2-O sulfation is important for migration, we analyzed 3T3 fibroblasts. Previously, we have shown that 3T3 fibroblasts express Ust (Nikolovska et al, 2014). Therefore, we knocked down Ust in 3T3 fibroblasts which led to a ,4063% reduction in Ust protein (Fig.…”

“…A solid-phase binding assay has previously shown that there were different binding properties between the highly sulfated CS/DS extracted from wild-type and Dcn 2/2 mice towards digoxygenin (Dig)-conjugated Fgf2 (Nikolovska et al, 2014). BSA was used as negative control and decorin with ,50% IdoUA content as positive control (Seidler at al., 2006).…”

“…Furthermore, the lack of CS/DS 2-O sulfation in skin of decorin-deficient (Dcn 2/2 ) mice affects Fgf2 and Fgf7 binding and keratinocyte differentiation (Nikolovska et al, 2014). For HS-Fgf2 interaction, L-IdoUA 2-O sulfation plays a vital role and influences binding of Fgf2 to the receptor (Jemth et al, 2002;Ashikari-Hada et al, 2009).…”