Hypophosphatasia is a heritable, untreatable, bone mineralization disease of variable clinical severity and pattern of inheritance caused by mutations that affect the tissue-nonspecific alkaline phosphatase (TNAP) gene. TNAP's primary function in bone is to hydrolyze inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. The elevated levels of PPi that accumulate in hypophosphatasia cause a secondary increase in the levels of osteopontin (OPN), another mineralization inhibitor, which likely contributes to the resulting rickets/osteomalacia characteristic of this disease. The genetic ablation of the molecules that produce and transport PPi to the extracellular space, i.e., NPP1 and ANK, lead to normalization of the extracellular PPi and OPN levels, resulting in the reversal of rickets/osteomalacia in the Akp2 (TNAP) knockout mice. In Aim 1 we will test the hypothesis that we can use chemical inhibitors to therapeutically target the function of NPP1 and ANK to cause normalization of both PPi and OPN levels and thus, achieve correction of the bone abnormalities of hypophosphatasia. We will also ascertain to what extent increased OPN levels contribute to the rickets/osteomalacia by examining the degree of correction that might be achieved in Akp2/OPN double deficient mice. We will also continue with our ongoing efforts to treat hypophosphatasia by cell/gene therapy. Elucidating the molecular basis of monomer-monomer crosstalk in TNAP heterodimers is crucial to our ability to understand and predict the severity of TNAP-mutant combinations and the mechanism(s) of pathogenesis, penetrance, expressivity and mode of inheritance for each mutation. In Specific Aim 2, we will clarify how each structural domain in the TNAP subunit contributes to the allosteric behavior of TNAP dimers and how they affect the kinetic properties of TNAP heterodimers towards the physiological substrates PLP, PPi and AMP. Also, since TNAP itself may be a useful therapeutic target to treat hvpermineralization disorders, we will elucidate the precise mechanism of TNAP inhibition to help us design more specific enzyme inhibitors for the clinical management of diseases such as ankylosis and osteoarthritis. Our work will provide fundamental information about the molecular mechanism(s) of pathogenesis of hypophosphatasia and the molecular basis for the different genetic modes of transmission. We also have a unique opportunity to develop successful treatments for hypophosphatasia and other bone diseases. [unreadable] [unreadable]