Polyamines are involved in the 1-α,25-dihydroxyvitamin D3-induced fusion of mouse alveolar macrophages

We have reported that 1-α,25-dihydroxyvitamin D3 [1-α,25-(OH)2D3] directly induces fusion of mouse alveolar macrophages at a very high rate (circa 70–80%) by a mechanism involving protein synthesis (Proc. Natl. Acad. Sci. USA 80:5583, 1983; FEBS Letters 174:61, 1984). While examining further the mechanism of the 1-α,25-(OH)2D3-induced fusion of macrophages, we found that polyamines are involved in this mechanism. Mouse alveolar macrophages incubated with 12 nM 1-α,25-(OH)2D3 began to fuse at 36 h and the fusion rate increased linearly up to 60 h. Addition of as much as 0.05–5 mM α-difluoromethylornithine (α-DFMO), a specific inhibitor of ornithine decarboxylase, did not inhibit fusion appreciably, but addition of 0.05–5 μM methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, strikingly inhibited fusion. When macrophages were treated with both 12 nM 1-α,25-(OH)2D3 and 5 μM MGBG for the first 12 h and incubated further for 60 h in fresh medium containing 1-α,25-(OH)2D3, fusion was significantly inhibited, suggesting that the 1-α,25-(OH)2D3-induced synthesis of polyamines precedes fusion. The inhibition by MGBG of the 1-α,25-(OH)2D3-induced fusion was restored completely by adding 1 μM spermidine or spermine or 100 μM putrescine. None of the polyamines alone induced fusion. MGBG suppressed the 1-α,25-(OH)2D3-induced incorporation of [3H]-leucine into the trichloroacetic acid-insoluble fraction in macrophages, but its inhibitory effect was restored completely by adding 1 μM spermidine. When macrophages were incubated with [14C]-ornithine, the polyamine that accumulated most was [14C]-spermidine. 1-α,25-(OH)2D3 further enhanced the accumulation of [14C]-spermidine. The accumulation of [14C]-putrescine and spermine was not appreciably altered by the 1-α,25-(OH)2D3 treatment. Adding MGBG almost completely suppressed the accumulation of [14C]-spermidine and spermine, but it enhanced the accumulation of [14C]-putrescine considerably. These results indicate that spermidine or spermine is an important intracellular mediator of the 1-α,25-(OH)2D3 action in inducing protein synthesis, which in turn somehow induces fusion of alveolar macrophages.