Molecular cloning and expression of a functionally different alternative splice variant of prointerleukin-1α from the rat testis

We report here the characterization of an alternative splice Variant of prointerleukin-1 alpha (proIL-1 alpha), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1 alpha: (32proIL-1 alpha) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1 alpha). In situ hybridization demonstrated constitutive expression of the splice transcript in the seminiferous tubules. This alternative complementary DNA lacked the fifth exon, harboring the calpain cleavage site essential for generation of mature 17K IL-1 alpha. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1 alpha, but not of 24proIL-1 alpha. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1 alpha to the mature 17K form and secretion, whereas 24proIL-1 alpha remained unprocessed. Both 32proIL-1 alpha: and 24proIL-1 alpha showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1 alpha. In contrast, when tested on hCG-stimulated Leydig cells in vitro, a dose-dependent inhibition of testosterone production was obtained with mature 17K IL-la and at a lower potency with 32proIL-1 alpha, whereas 24proIL-1 alpha was inactive. In conclusion, the three IL-I bioactive proteins described here contribute to IL-1 protein heterogeneity and may serve as constitutive paracrine mediators in the testis.