Post-transcriptional regulation of gene Expression during bone regeneration.

Abstract Co-transcriptional and post-transcriptional mechanisms play central roles in regulating gene expression. Understanding how these mechanisms control gene expression and, thus, cellular homeostasis is essential for the development of novel therapeutic approaches. Splicing is a co-transcriptional process during which introns are removed from pre-mRNAs and the remaining exons are connected. Connecting the exons in different combinations is called alternative splicing (AS), a process that generates diversified mature mRNAs from the same gene. AS plays crucial roles in regulating gene expression, and mis-splicing is implicated in different diseases. Thus, AS is a tightly regulated process. In spite of its well-established roles, AS is understudied in the context of fracture healing. Proteoglycan 4 (PRG4) is an extracellular matrix protein found throughout the animal kingdom and is best known for its lubricant effects in articular cartilage. PRG4 is highly expressed in the synovial fluid and possesses chondroprotective effects; therefore, exogenous expression of recombinant PRG4 in the articular cartilage halts the progression of osteoarthritis. PRG4 is also expressed in organs other than articular joints, including the liver, heart, and bone. However, the biological functions of PRG4 in these tissues are not well defined. Importantly, the expression pattern and biological functions of PRG4 during fracture healing have never been characterized. Our preliminary data indicate high expression of Prg4 in the callus periosteal stem cells (PSC) during the inflammatory phase of fracture healing. Interestingly, Prg4 is predominantly expressed in callus PSC as a splice variant that lacks 3 coding exons and has bever been reported in any musculoskeletal tissue. This splice variant is conserved in human cells. TGFB1 significantly induces Prg4 expression and reprograms Prg4 splicing, which makes Prg4 expression and splicing pattern in the callus PSC distinct from those in the intact- bone PSC. Inhibiting the expression of Prg4 in the callus results in reduced abundance of PSC, defective soft- callus formation, and reduced callus mineralization and bone formation. Thus, we hypothesize that Prg4 undergoes a unique splicing pattern in the callus that is mediated, at least in part, by TGFB1, resulting in a splice variant that plays critical autocrine and paracrine regulatory roles and impacts different healing phases. In Aim 1, we will assess the roles of the Prg4 splice variant in different healing phases and in maintaining the homeostasis of different cell types in the callus. In Aim 2, we will study the autocrine effects of PRG4 and how it regulates the homeostasis and differentiation of PSC. In Aim 3, we will identify the molecular pathways that regulate Prg4 expression and splicing, define the unique roles of different splice variants of Prg4 in regulating PSC and human BMSC homeostasis, and determine the receptors via which Prg4 signals. Completion of our studies will bridge several knowledge gaps and identify novel roles of PRG4 in bone repair.