Molecular cloning and expression of a functionally different alternative splice variant of prointerleukin-1alpha from the rat testis.
We report here the characterization of an alternative splice variant of prointerleukin-1alpha (proIL-1alpha), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1alpha (32proIL-1alpha) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1alpha). 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-1alpha. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1alpha, but not of 24proIL-1alpha. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1alpha to the mature 17K form and secretion, whereas 24proIL-1alpha remained unprocessed. Both 32proIL-1alpha and 24proIL-1alpha showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1alpha. In contrast, when tested on hCG-stimulated Leydig cells in vitro, a dose-dependent inhibition of testosterone production was obtained with mature 17K IL-1alpha and at a lower potency with 32proIL-1alpha, whereas 24proIL-1alpha was inactive. In conclusion, the three IL-1 bioactive proteins described here contribute to IL-1 protein heterogeneity and may serve as constitutive paracrine mediators in the testis.
Sultana
T
T
Department of Woman and Child Health, Karolinska Institute and Hospital, Stockholm, Sweden.
Svechnikov
K
K
Weber
G
G
Söder
O
O
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002154:Calpain / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005091:Exons; D015870:Gene Expression; D017403:In Situ Hybridization; D007375:Interleukin-1 / Q000737:chemistry / Q000235:genetics*; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013737:Testis / Q000737:chemistry*; D013739:Testosterone / Q000096:biosynthesis; D014162:Transfection
D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D007375:Interleukin-1; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013739:Testosterone; D002154:Calpain