Infantile hypophosphatasia: Enzymatic defect explored with alkaline phosphatase-deficient skin fibroblasts in culture

Summary

Evidence that infantile hypophosphatasia may result from defective regulation of an intact structural gene for the tissue nonspecific (bone/liver/kidney) isoenzyme of alkaline phosphatase (TNSALP) was explored by studying physicochemical properties of ALP in sonicates of monolayers of cultured dermal fibroblasts from 7 patients (PT) and 5 age- and sex-matched control (CT) subjects. Both groups had low levels of ALP activity when assayed with 4-methylumbelliferyl phosphate substrate. The mean specific activity of ALP in the PT fibroblasts was markedly subnormal (V_max less than 1% of CT), but apparently not from extracellular loss of enzyme, since defined medium had less ALP activity when conditioned by PT compared to CT cells. Although the mean Km for the sonicate ALP was similar for both groups at pH 10.1, pH optimum, thermal stability and response to several inhibitors appeared to be different. Nevertheless, it seemed that some TNSALP-like enzyme was present in the PT group. Exposure of cells in culture to 5-azacytidine and several putative inducers of ALP failed to increase the enzyme activity in either the PT or CT groups. Had the physicochemical properties of the constitutive (or inducible) ALP been the same in the PT and CT cell groups, the findings would have provided evidence for the generality of our previous observations in one patient which indicated that defective regulation of an intact structural gene for TNSALP could account for hypophosphatasia. Our results do not, however, refute this possibility; e.g., especially minute amounts of typical TNSALP may have been present in our PT fibroblasts but were masked by (1) normal or compensatory expression of trace levels of ALP from a separate gene, (2) heterogeneity of the ALPs in the various PT (or CT) cell lines, or (3) the presence of some additional TNSALP that was different from the constitutive ALP of normal fibroblasts. Indeed, review of the findings for individual cell lines suggested that in one patient with relatively high levels of ALP activity, the physicochemical properties of this ALP was most like the enzyme in CT cells. Although further studies—including use of sensitive immunologic and electrophoretic methods—will be necessary to further characterize the minute amounts of residual ALP(s) in infantile hypophosphatasia tissues, presence of some TNSALP-like ALP in PT fibroblasts indicates that the disorder is not commonly due to a complete gene deletion for TNSALP. Our findings do indicate an especially profound and stable deficiency of typical constitutive TNSALP in fibroblasts in this disorder and suggest, therefore, that these cells might be a valuable model to explore the physiologic role of TNSALP.