OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine the mechanism of action of myostatin on osteoblasts by measuring markers of osteoblast differentiation. With these experiments we will evaluate the effects of myostatin on an osteoblastic cell line (MC3T3 cells) and primary murine osteoblasts during baseline and hyperglycemic conditions and assess whether these effects are altered in the presence of a hyperglycemic environment. METHODS/STUDY POPULATION: Primary osteoblasts from calvaria of WT mice will be isolated and cultured per previously published protocol. MC3T3 cells (murine pre-osteoblast cell line) and primary osteoblasts will be plated in 6-well plates until they reach confluency. They will subsequently be stimulated with or without myostatin at various concentrations under control and hyperglycemic conditions. Additional experiments will assess myostatin stimulation during cell differentiation/maturation in the presence of osteogenic induction medium. Subsequently, cells will be lysed and processed for gene analysis with qPCR. Genes of interest (e.g., myostatin, RUNX2, osteocalcin etc.) will be assessed. Additionally, cells will be collected and processed for protein quantification with western blot to assess myostatin-related pathways, such as Smad2/3 and MAPK signaling. RESULTS/ANTICIPATED RESULTS: We have demonstrated that the receptor for myostatin (Activin receptor 2b, AcvR2b) is present in MC3T3 cells and we have evidence of Smad2 phosphorylation in MC3T3 cells as a result of myostatin stimulation, confirming that myostatin can exert intracellular signaling events in bone cells (Fig 1). We anticipate to observe negative effects of myostatin on differentiation of primary osteoblasts and MC3T3 cells. Specifically, we anticipate suppression of Runt-related transcription factor 2 (RUNX-2), a transcription factor known as the “ master regulator” of osteogenic gene expression and programming, as a result of signaling downstream of Smad 2/3. Additionally we anticipate downregulation of osterix and osteocalcin, two essential genes for osteoblast differentiation and activity. We anticipate that hyperglycemia will potentiate the negative effects of myostatin on osteoblastogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: We have demonstrated that myostatin can directly act on osteoblastic cells. As myostatin is a negative regulator or bone mass, its direct effects on bone cells can be detrimental to the bone health of patients with elevated myostatin levels and/or activity. There is evidence suggesting that myostatin is elevated in Type 1 diabetes, and its effects might be potentiated in a hyperglycemic environments. Future experiments will be evaluating the role of myostatin on a diabetic animal model and in humans. Our experiments provide an additional mechanism by which muscle-bone interactions could be contributing to the development of diabetic bone disease.