Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

Abstract

We have previously shown that the signal peptideless cytokine interleukin 1a (IL-la) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249: [1570][1571][1572][1573][1574] 1990). To investigate the potential intracellular function of IL-lot, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-la, the precursor molecule IL-11-27, and the mature protein IL-11.3-271* The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-la gene and the P-galactosidase open reading frames. The IL-1 113-271 protein was cytoplasmic, while IL-11-271 was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-lot nuclear targeting. Moreover, nuclear localization of IL-la correlates with impaired cell growth and expression of some IL-la-inducible genes. These results suggest that transport of endogenous intracellular IL-lat has been highlighted by the discovery of an intracellular IL-1 receptor antagonist which is expressed in keratinocytes (17). This protein could function as an intracellular competitor of either internalized or endogenously produced IL-la.To determine whether IL-lat represents an intracellular regulatory molecule in endothelial cells, we transfected transformed endothelial cells (tEC) (46) with plasmids containing the cDNAs that code for IL-11-271 and IL-1 113-271 The subcellular localization of the two forms was investigated directly or by using chimeric genes constructed by fusion of the IL-lot and ,B-galactosidase (P-Gal) open reading frames. IL-11_271 was nuclear, while IL-11,3-271 was cytoplasmic. The sequence responsible for the nuclear targeting of IL-1 is contained within seven amino acid residues at positions 79 to 85. We also determined whether or not the activity of endogenous IL-la correlates with its ability to localize to the nucleus. The following data suggest that transport of IL-la into the nucleus is required for it to modulate endothelial cell properties. MATERIALS AND METHODSPlasmid construction. Most plasmids were constructed by PCR amplification of the IL-la cDNA with primers bearing unique sequence and restriction enzyme sites. Plasmid Cfla, containing the full-length cDNA for IL-lat, was kindly provided by R. Gayle (Immunex, Seattle, Wash.).The primers used to clone IL-lot in pMEXneo (29) were IL-11-271 sense (5'-CCG TCG ACC CAC CAT GGC-3'),

The precursor form of IL-1α is an intracrine proinflammatory activator of transcription

“…As an intracellular molecule, IL-1␣ has been also shown to be inserted in the membrane and act in a juxtacrine manner (3,4,23). However, there is growing evidence for intracellular functions of IL-1␣, including promotion of senesence (12), cell growth and differentiation (7) and regulation of gene expression (24).…”

“…pIL-1␣ possesses a KVLKKRR nuclear localization site in its N-terminal propiece (6). In fact, pIL-1␣ translocates to the nucleus in a variety of cells, and this appears to be necessary for specific downstream events (7). pIL-1␣ is found in keratinocytes and epithelial cells of healthy subjects, and mice deficient in IL-1␣ do not exhibit an apparent phenotype (8) unless challenged by disease processes, suggesting that a putative function for the intracellular pIL-1␣ requires an additional external stimulus.…”

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) delays and induces escape from senescence in human dermal microvascular endothelial cells

“…However, PD45 cells cultured with VPF/VEGF continuously after PD26 and then withdrawn from VPF/VEGF for 7 days incorporated signi®cantly less thymidine than PD47 cells cultured continuously with VPF/VEGF (P50.001) therefore, the p53 expressed in senescent cells and VPF/VEGF-treated cells could be functionally different, a possibility that requires further investigation. Maier et al (1994) suggested that endogenous IL-1a is critical in the progression of human umbilical vein endothelial cells toward senescence. However, we did not detect any signi®cant di erences in IL-1a expression between young, senescent and VPF/VEGF-treated HDMEC (data not shown).…”

Genomic profiling of C/EBPβ2 transformed mammary epithelial cells: A role for nuclear interleukin-1β

“…IL1ra binds interleukin-1 receptor 1 (IL1R1), and prevents IL1b mediated signal transduction. 18 Even at 1,000 fold molar excess, IL1ra had no effect on the growth of MCF10A-C/EBPb2 cells, either in the presence or absence of EGF (Fig. 2F).…”