Intracellular Precursor Interleukin (IL)-1α, but Not Mature IL-1α, Is Able to Regulate Human Endothelial Cell Migration in Vitro

Abstract

The human umbilical vein endothelial cell (HUVEC) has a finite lifespan in vitro, and senescent HUVEC contain elevated levels of the negative growth regulator interleukin (IL)-1␣. IL-1␣ is translated as a signal peptide sequence-less cytosolic 31-kDa precursor (IL-1␣ p), which undergoes proteolytic activation to release the mature carboxyl terminus 17-kDa protein (IL-1␣ m). Both the IL-1␣ p and IL-1␣ m proteins are biologically active as exogenous cytokines. Interestingly, only IL-1␣ p contains a nuclear localization sequence between residues 79 and 85. To further study the role of intracellular IL-1␣ in the regulation of human endothelial cell function, a spontaneous HUVEC transformant was stably transfected with IL-1␣ p, IL-1␣ m, and the IL-1␣ p K82N mutant, which attenuates the nuclear traffic of IL-1␣ p. Interestingly, the IL-1␣ p transfectants were found to have a lower migratory potential than either IL-1␣ m or IL-1␣ p K82N transfectants, and the addition of the IL-1 receptor antagonist did not alter the migration of these cells. Immunofluorescence microscopy demonstrated that only the IL-1␣ p transfectants exhibited prominent staining for ␤-catenin-associated cell-to-cell contacts, as well as pronounced vimentin intermediate filaments and actin cytoskeleton staining. These data suggest that IL-1␣ p, and not IL-1␣ m, may function as an intracellular regulator of the migratory capacity of the human endothelial cell and that the nuclear localization sequence present within IL-1␣ p may be involved in regulating this function. Interleukin (IL)1 -1 consists of a family of cytokines which play an important role in inflammation and the response to injury (1). The family consists of three members, IL-1␣, IL-1␤, and the IL-1 receptor antagonist protein (IRAP). Signal transduction is initiated by the binding of either IL-1␣ or IL-1␤ to its receptor; conversely, as its name suggests, the binding of IRAP to the IL-1 receptor does not activate any downstream effector molecules, but rather, acts as a competitive inhibitor of ligandreceptor binding (2).The IL-1 family is expressed as precursor proteins; the precursors of IL-1␣ and IL-1␤ are translated as 31-kDa proteins, which are processed to the mature carboxyl terminus 17-kDa forms (3). A calpain-like protease cleaves IL-1␣ between residues 112 and 113 (4); however, expression of either the precursor (p; IL-1␣ p) or mature (m; IL-1␣ m) forms of the protein in a rabbit reticulocyte system has shown that both IL-1␣ p and IL-1␣ m bind to the IL-1 receptor and are biologically active (5, 6). Conversely, IL-1␤ is only functional as the 17-kDa protein, with proteolytic activation by the IL-1␤-converting enzyme being necessary for its activity (7). Interestingly, the 16-kDa amino-terminal domain of the IL-1␣ p contains a nuclear localization signal (NLS) (8) and is translocated to the nucleus and produces a transformed phenotype when expressed in rat mesangial cells (9).Activation of monocytes by lipopolysaccharide or phorbol myristic acid leads to the release of IL-1␣ p and...

Intracellular Interleukin-1α Functionally Interacts with Histone Acetyltransferase Complexes

“…The original vector contains Myc and His tags downstream of the cloned cDNA, therefore, all the IL-1␣ proteins were expressed as double tagged chimeras with N-terminal FLAG and C-terminal Myc and His tags. Fragments encoding human deletion mutants IL-1NTP-VVATN (internal deletion aa 78 -87), IL-1NTP-EDSSS (terminal deletion aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], and IL-1NTP-ITDDD (terminal deletion aa 96 -111) were amplified and cloned into BamHI and XhoI sites of modified pcDNA4/TO/MycHis (see above) vector.…”

“…Indeed, overexpressed intracellular IL-1␣ precursor (pre-IL-1␣), but not IL-1MAT, was able to inhibit cell growth and to induce the expression of the plasminogen activator inhibitor-1 and collagenase genes (9,10). Pre-IL-1␣ was shown to stimulate proliferation of smooth muscle cells (11) and to regulate the migration and the lifespan of endothelial cells (12,13). Thus, various effects of pre-IL-1␣ are found to be dependent on the presence of the acidic IL-1NTP fragment with the predicted pI 5.1.…”

The non-classical export routes: FGF1 and IL-1α point the way

“…Since these studies suggested that TTM can repress the transcriptional activation of NF-κB, and NF-κB lies downstream of IL-1 receptor signaling (Baldwin, 1996), it appeared possible that TTM functions as a repressor of nonclassical IL-1α export. Both FGF1 and IL-1 play a proangiogenic role in vivo (Friesel and Maciag, 1999;Voronov et al, 2003) although, in vitro, FGF1 stimulates proliferation and migration of endothelial cells (Maciag et al, 1979;McMahon et al, 1997), whereas IL-1α inhibits both of these activities ). It appears that the regulation of angiogenesis and inflammation involves a coordination of nonclassical FGF1 and IL-1α release.…”

Membrane-Associated IL-1 Contributes to Chronic Synovitis and Cartilage Destruction in Human IL-1α Transgenic Mice

“…As the transgene includes a nuclear localization sequence (aa 79 -86) that has been shown to be important for IL-1␣ association with the plasma membrane (24), MA-IL-1 is expected to express in Tg mice and play an important role in the development of joint destruction. The present study investigated whether biologically active hIL-1␣ derived from the transgene appears on the surface of synoviocytes, and whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis in hIL-1␣ Tg mice.…”