The broad objectives of this proposal are to determine how proteoglycan 4/lubricin regulates anabolic, catabolic and inflammatory signaling networks in equine articular tissues to protect against joint injury. Evidence is mounting to suggest that lubricin, in addition to its known boundary lubricating and anti-adhesive properties, is able to modulate immune responses and inflammatory signaling pathways to maintain tissue homeostasis after joint injury. However, little is known about how lubricin performs these functions and whether these responses differ when proteoglycan 4 (PRG4) is genetically overexpressed or administered as a recombinant glycoprotein.

To investigate these questions, PRG4 will be overexpressed in equine primary cell lines using a transposasebased vector for stable genome editing, and recombinant equine lubricin will be produced in HEK293F cells. We have recently reported upon a codon scrambling, custom gene synthesis approach that enabled efficient production of full-length recombinant human lubricin and other mucinous glycoproteins, which will be applied to recombinant equine lubricin. Transcriptomic analysis will be performed using RNA-Seq and validated with RTqPCR. In vitro assessment of recombinant equine lubricin administration to equine primary cell lines and joint tissues will include evaluation of cell growth and adhesion; anabolic and catabolic activity using biochemical assays; and transcriptome regulation using RNA-Seq and RT-qPCR. The ability of recombinant equine lubricin to ameliorate IL-1-induced carpal synovitis will be evaluated in vivo using clinical, cytologic and biochemical parameters, and safety will be evaluated through histologic, gene expression and biochemical analyses in naïve joints injected with recombinant equine lubricin.

Specific Aim 1. To identify transcriptional networks regulated by proteoglycan 4 (PRG4) expression in equine synoviocytes, chondrocytes and mesenchymal stromal cells (MSCs).

Specific Aim 2. To investigate how recombinant equine lubricin regulates cell growth and adhesion, gene expression, and biochemical parameters in equine primary cell lines and explant cultures.

Specific Aim 3. To determine whether recombinant equine lubricin can modulate clinical, cytologic or biochemical parameters following intra-articular injection in IL-1-induced carpal synovitis, and to demonstrate safety and absence of adverse effects following lubricin injections into naïve fetlock joints.