Continuous activation of gpl 30 , a signal - transducing receptor component for interleukin 6 - related cytokines , causes myocardial hypertrophy in mice ( interleukin 6 receptor )

To investigate the physiological roles of gpl30 in detail and to determine the pathological consequence of abnormal activation of gpl30, transgenic mice having continuously activated gpl30 were created. This was carried out by mating mice from interleukin 6 (IL-6) and IL-6 receptor (IL-6R) transgenic lines. Offspring overexpressing both IL-6 and IL-6R showed constitutive tyrosine phosphorylation of gpl30 and a downstream signaling molecule, acute phase response factor/signal transducer and activator of transcription 3. Surprisingly, the distinguishing feature of such offspring was hypertrophy of ventricular myocardium and consequent thickened ventricular walls of the heart, where gpl30 is also expressed, in adulthood. Transgenic mice overexpressing either IL-6 or IL-6R alone did not show detectable myocardial abnormalities. Neonatal heart muscle cells from normal mice, when cultured in vitro, enlarged in response to a combination ofIL-6 and a soluble form of IL-6R. The results suggest that activation of the gpl30 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in physiological regulation of myocardium. gpl30 was initially identified as a signal-transducing receptor component that associates with the interleukin 6 receptor (IL-6R) when the receptor is occupied with interleukin 6 (IL-6). It has been revealed that the receptor complexes for IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, and ciliary neurotrophic factor (CNTF) utilize this gpl30 protein as a common signal-transducing component (1-10). From the study on cytokine receptor complexes sharing gpl30, it is now clear that a general first step in the signaling processes of cytokines appears to be ligand-induced dimerization of receptor components whose cytoplasmic regions interact to activate downstream molecules (2, 5). These molecules include members of a JAK family of nonreceptor tyrosine kinases, JAK1, JAK2, and TYK2, and a latent cytoplasmic transcription factor, acute phase response factor/signal transducer and activator of transcription 3 (APRF/STAT3) (11-16). In the case of the IL-6R system, a complex of IL-6 and IL-6R [either in a membrane-anchored form or in an extracellular soluble (s) form] associates with gpl30 to induce its homodimerization (1, 17, 18). Thus, the addition of sIL-6R to IL-6-responsive cells, whose phenotype is IL-6R+/gp130+, enhances their responsiveness to IL-6. The IL-6-sIL-6R complex, when added to IL-6R-/gpl30+ cells, which normally are nonresponsive to IL-6, confers their responsiveness to IL-6. This complex has been observed to mimic the actions of not only IL-6 but also IL-11, LIF, oncostatin M, and CNTF at least in vitro in cells expressing gp130 but not cytokine-specific receptor molecules (19, 20). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. gpl3O is ubiquitously expressed in almost all the tissues examined (21), whereas the cytokine-specific receptors show a somewhat restricted distribution. Since the functions of gpl3O stimulatory cytokines have been studied in greater detail in vitro, physiological functions of gpl3O are not considered to have been fully elucidated. To investigate detailed functions of gpl3O in vivo and to determine the pathological consequence of abnormal activation of gpl3O, transgenic mice with continuously activated gpl3O protein have been made. Two transgenic lines were separately prepared by introducing minigenes of human (h) IL-6 and membrane-anchored hIL-6R under promoters constitutively switched on in immune (murine major histocompatibility complex class I H-2Ld promoter) (22) and ubiquitous cell systems (chicken f3-actin promoter) (23), respectively. These two transgenic lines were mated to obtain "double-transgenic" mice expressing both hIL-6 and hIL-6R proteins, in which gpl3O protein is continuously activated. We here describe the physiological and pathological roles of gpl3O in the double-transgenic mice by focusing our attention on the myocardial system. MATERIALS AND METHODS Transgene Construction and Production of Transgenic Mice. The IL-6R transgene was prepared by inserting an 1.7-kb fragment of hIL-6R cDNA (24) into a unique Xho I site of pCAGGS (23), which carries the chicken f3-actin gene promoter. This vector was then digested with Sac I and HindIll to generate a linear fragment and microinjected into the pronuclei of fertilized BDF1 mouse eggs. Mice were obtained as described (25). The generation of IL-6 transgenic mice has been described elsewhere (22). Determination of shIL-6R. Serum levels of shIL-6R were measured by ELISA (26) with anti-hIL-6R monoclonal antibody (Ab) MT18 (27). Immunoblot Analysis. Heart cells were solubilized with Nonidet P-40 lysis buffer, and clear lysates obtained by centrifugation were incubated with anti-gpl30 monoclonal Ab and anti-APRF/STAT3 polyclonal Ab (12). Immunoprecipitates using protein A-Sepharose (Pharmacia) were analyzed by SDS/PAGE and subsequent immunoblotting with anti-gpl30 polyclonal Ab (19), anti-APRF/STAT3 polyclonal Ab, and anti-phosphotyrosine Ab (4G10; Upstate Biotechnology, Lake Placid, NY) using an enhanced chemiluminescence (ECL) detection system (Amersham) according to the manufacturer's procedures. Histology. Samples were fixed in 3.7% (wt/vol) formaldehyde in phosphate-buffered saline and processed for paraffin Abbreviations: IL-6, interleukin 6; IL-6R, IL-6 receptor; s, soluble; h, human; LIF, leukemia inhibitory factor; CNTF, ciliary neurotrophic factor; APRF/STAT3, acute phase response factor/signal transducer and activator of transcription 3; Ab, antibody.