Molecular and functional studies of interleukin-1a in the rat testis

Sammanfattning: The aim of the present study was to gain a better insight into the role of interleukin-1 alpha (IL-1 alpha) in testicular physiology. In testis, IL-1alpha acts as a paracrine mediator involved in the regulation of testicular functions. Bioactive IL-1 alpha isolated and characterized from adult rat testis, showed the presence of several distinct protein species, with a size range of 17-40 kDa and charge heterogeneity. The present study was undertaken to clarify the molecular heterogeneity and to characterize different isoforms of IL-1alpha in rat testis and to study the processing, secretion, regulation and function of these isoforms. The physicochemical characterization revealed that testicular IL-1 alpha is similar to IL-1 alpha from activated macrophages. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1 alpha antiserum, IL-1 receptor antagonist and soluble type 1 IL-1 receptor. Three isoforms of IL-1 alpha proteins with molecular sizes of 45, 31 and 17 kDa and at charges of pH 5.7 and 6.0 are found in the testis. All three forms are secreted into the interstitial compartment and tubular lumen. Molecular characterization revealed that heterogeneity also existed at the transcriptional level and a novel splice variant was found and named as 24proIL-1 alpha. It lacked exon 5, which harbors the cleavage site of the processing enzyme calpain. Expression of these isoforms in COS. cells and in vitro calpain cleavage assay of recombinant protein expressed in E. coli confirmed that 32proI1l-1 alpha was processed to produce 17 kDa whereas 24proIL- 1 alpha was resistant to cleavage. Both forms were bioactive in thymocyte proliferation IL-1 bioassay. However, these isoforms showed differential activity on human chorionic gonadotropin (hCG)-induced Leydig cell steroidogenesis. 32proIL-1 alpha was inhibitory and 24proIL-1 alpha showed no effect. On analysis of mechanism of action, 24proIL- 1alpha showed weak but significant stimulation of hCG-induced steroidogenesis in Leydig cells from 40- day-old rats, while 32proIL-1 alpha was inhibitory. All IL-1 isoforms showed stimulation of basal testosterone production in Leydig cell from 40-day-old rats but not 80-dayold rats. These effects were receptor-mediated, and protein kinase A and Ca 2+ seemed to be involved as major components of the IL- 1 alpha signaling cascade. IL-1 alpha isoforms stimulated proliferation of T lymphocytes and growth of immature Sertoli cells. All isoforms were active in both growth assays and showed differential biopotencies in the following order: 17 kDa IL-1 alpha > 32proIL-1 alpha > 24proIL-1 alpha. On analysis of the subcellular localization of IL-1 alpha isoforms, COS-1 and CHO showed differential subcellular localization of these isoforms, where 24proIL-1 alpha in CHO cells showed granular appearance. The MSC-1 Sertoli cell line expressed IL- 1 alpha constitutively. The regulation of IL-1 alpha was studied at the level of the enzyme calpain, the key regulator of production of the mature 17 kDa form of IL-1 alpha from 32proIL-1alpha. Both calpains (1 and 11) cleaved recombinant 32proIL-1alpha in vitro. In response to LPS, calpain I protein levels were downregulated in seminiferous tubules whereas calpain II was less affected. By contrast, liver after LPS treatment showed up-regulated expression of calpain I and II. In summary, the study showed that testicular IL-1 alpha exists in three isoforms that are secreted into the tubular lumen and interstitial space. These isoforms showed bioactivity with differential biopotencies. Thus IL-1 alpha acts as a paracrine factor in adult rat testis.