Circulating fibroblast growth factor-like substance in familial multiple endocrine neoplasia type 1.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. Basic FGF lacks a classical signal sequence, and it is not clear how it is released from cells. bFGF or bFGF-like activity has not been previously demonstrated in plasma. In an earlier study we showed increased mitogenic activity for parathyroid-derived epithelial and mesenchymal cells in plasma of subjects with familial multiple endocrine neoplasia type 1 (FMEN1). In the present study we examined the growth-promoting activity of normal and FMEN1 plasmas [applied to heparin-Sepharose (HS) columns] in parathyroid-derived cloned endothelial cells. FMEN1 plasma HS-adsorbed activity exceeded normal plasma HS-adsorbed activity in 6 of 8 FMEN1 plasma samples. Peak (FMEN1 plasma) HS-adsorbed activity eluted with 0.1-0.3 M NaCl, was completely neutralized by specific antibodies against bFGF, and had an apparent mol wt of 110 kD. Active fractions from FMEN1 plasma prepared by gel filtration in 7 M urea displayed apparent mol wt of about 14-16 kD and showed increased apparent affinity for HS; recovered activity appeared principally in the 3.0-M NaCl eluate. Using a sensitive two-site immunoradiometric assay for bFGF we found 0.4 ng/mL bFGF-like immunoreactivity in the highly purified 3.0-M NaCl eluate from a HS column to which the active components from gel filtration of FMEN1 plasma in 7 M urea were applied. These results imply that bFGF or closely related factors circulate in FMEN1.

Selective expression of a normal action of the 1,25-dihydroxyvitamin D3 receptor in human skin fibroblasts with hereditary severe defects in multiple actions of that receptor.

We evaluated three actions of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in human skin fibroblasts to test for heterogeneity in hormone-response coupling. In fibroblasts from normal subjects the 1,25-(OH)2D3 concentrations for half-maximal effect (EC50) were: for mitogenic effect 0.0001-0.0005 nM, for antimitogenic effect 1 nM, and for induction of 25-OHD3 24-hydroxylase (24-OHase) 5 nM. To evaluate the effects of mutations presumed to be in the gene for the 1,25-(OH)2D3 receptor we examined cell lines representing four kindreds with hereditary resistance to 1,25-(OH)2D3 ("mutant" cell lines). In one mutant cell line all three 1,25-(OH)2D3 actions were severely abnormal. In one mutant cell line 24-OHase induction and mitogenic action were undetectable, but EC50 and maximal effect were normal for antimitogenic action of 1,25-(OH)2D3. In two mutant cell lines 24-OHase induction and antimitogenic actions were undetectable or severely impaired but mitogenic action were undetectable or severely impaired but mitogenic action was normal in EC50 and normal or increased in maximal effect. The mitogenic and antimitogenic actions in normal cells showed a similar profile of potency ratios for 1,25-(OH)2D3 and six analogues. Whenever a mutant cell showed a normal or even an abnormal mitogenic or antimitogenic effect of 1,25-(OH)2D3, these effects showed potency ratios similar to wild type, suggesting mediation by a similar 1,25-(OH)2D3 receptor. We conclude that three 1,25-(OH)2D3 actions show important differences in hormone response coupling indicated by differences in EC50 for 1,25-(OH)2D3 and by different consequences of receptor mutations.

Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts.

UNLABELLED: 1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. IN CONCLUSION: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.