1 Tissue - nonspecific alkaline phosphatase ( TNAP ) as a target of 2 sFRP 2 in cardiac fibroblasts 3

23 24 25 Recent studies of myocardial infarction in secreted Frizzled-related protein 2 (sFRP2) 26 knockout mice and our hamster heart failure therapy based on sFRP2 blockade have 27 established sFRP2 as a key profibrotic cytokine in the heart. The failing hamster heart is marked 28 by prominent fibrosis and calcification with elevated expression of sFRP2. Noting the 29 involvement of tissue-nonspecific alkaline phosphatase (TNAP) in bone mineralization and 30 vascular calcification, we determined whether sFRP2 might be an upstream regulator of TNAP. 31 Biochemical assays revealed a ~2-fold increase in the activity of TNAP and elevated levels of 32 inorganic phosphate (Pi) in the failing heart compared to the normal heart. Neither was this 33 change detected in the liver or hamstring muscle nor was it associated with systemic 34 hyperphosphatemia. TNAP was readily cloned from the hamster heart and upon overexpression 35 increased the level of extracellular but not intracellular Pi, which is consistent with the cell 36 surface location of the ectoenzyme. In line with the previous demonstration that sFRP2 37 blockade attenuated fibrosis, we show here that the therapy downregulated TNAP. This in vivo 38 finding is corroborated by the in vitro study showing that cultured cardiac fibroblasts treated with 39 recombinant sFRP2 protein exhibited progressive increase in the expression and activity of 40 TNAP, which was completely abrogated by cycloheximide or tunicamycin. Induction of TNAP by 41 sFRP2 is restricted to cardiac fibroblasts among the multiple cell types examined, and was not 42 observed with sFRP4. The current work indicates that sFRP2 may promote cardiac 43 fibrocalcification through coordinate activation of Tolloid-like metalloproteinases and TNAP. 44