Effect of Versican G1 Domain Overexpression on Hyaluronan, Hyaluronidase-2, and Synovial Joint Formation in the Embryonic Chick
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Date
2013
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Authors
Andrews, Kristen Nichole
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East Carolina University
Abstract
Versican, an extracellular chondroitin sulfate proteoglycan which binds hyaluronan, has been hypothesized to play a role in synovial joint formation in the chick embryo. Several studies have shown versican to be involved in mesenchymal aggregation during limb chondrogenesis with diminished expression as later stages of limb development occur. Although expression within the cartilage model is decreased, versican is still found in a few areas including the joint interzone and perichondrium. The presence of versican along with hyaluronan in the interzone could further joint interzone formation, the process of which is still not completely understood. Previous studies showed that the hyaluronan degratory enzyme, hyaluronidase 2 (Hyal-2), was up-regulated in the developing joint by overexpression of the hyaluronan-binding G1 domain of versican, suggesting a role for versican in regulation of hyaluronan metabolism during early stages of synovial joint morphogenesis. The goal of this study was to manipulate versican and hyaluronan expression in the embryonic chick to obtain a better understanding of their interaction during synovial joint development. To study versican's function, an adenovirally-encoded G1 N-terminal hyaluronan-binding domain was over-expressed via microinjection into the presumptive elbow joint of embryonic chicks. To examine the potential interaction of versican with the hyaluronan pathway during joint development, microinjection of dominant negative CD44 adenovirus, the hyaluronan synthesis inhibitor 4-methylumbelliferone, and two hyaluronidase2 shRNA viruses (hyal2-702 and hyal2-1130) with or without co-injection of adeno-G1 versican was performed. After injection, changes in expression of hyaluronan were observed via histochemistry at early cavitation stages (HH35/36). Morphological changes in the developing joint were observed using whole mount Alcian blue histochemistry and histochemical/immunocytochemical examination of tissue sections. To investigate whether adeno-G1 versican effects were dependent on hyaluronan or CD44, hyaluronan ELISA was performed to assess its accumulation due to up-regulation of Hyal-2. Overexpression of versican G1 in the developing synovial joint led to an enlargement of the interzone area and increased hyaluronan staining was seen in areas of adeno-G1 infection within treated limbs in comparison to controls. Treatment with 4-methylumbelliferone in conjunction with adeno-G1 also resulted in an increased interzone area but an overall decrease in hyaluronan accumulation. Hyaluronan ELISA results demonstrated reduced hyaluronan levels in the 4-methylumbelliferone treated samples in comparison to adeno-G1 alone. Immunohistochemical/histochemical staining along with trends seen from real-time PCR analysis of Hyal-2 suggests that the ability of G1 versican overexpression to upregulate Hyal-2 is dependent on hyaluronan.