Tài liệu Báo cáo Y học: Differential response of neuronal cells to a fusion protein of ciliary

Differential response of neuronal cells to a fusion protein of ciliary

neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory

factor

Pia Ma¨ rz1,*, Suat O¨ zbek2,*, Martina Fischer3

, Nicole Voltz4

, Uwe Otten1 and Stefan Rose-John4,5

1

Department of Physiology, University of Basel, Switzerland; 2

Department of Biophysical Chemistry, Biocenter, University of Basel,

Switzerland; 3

Xerion Pharmaceuticals, Martinsried, Germany; 4

Department of Medicine, Section Pathophysiology, Johannes

Gutenberg University of Mainz, Germany; 5

Department of Biochemistry, Christian Albrechts University of Kiel, Germany

Ciliary neurotrophic factor (CNTF) displays neurotrophic

activities on motor neurons and neural cell populations both

in vivo and in vitro. On target cells lacking intrinsic expression

of specific receptor a subunits cytokines of the IL-6 family

only act in the presence of their specific agonistic soluble

receptors. Here, we report the construction and expression of

a CNTF/soluble CNTF-receptor (sCNTF-R) fusion protein

(Hyper-CNTF) with enhanced biological activity on cells

expressing gp130 and leukemia inhibitory factor receptor

(LIF-R), but not membrane-bound CNTF-R. At the cDNA

level, the C-terminus of the extracellular domain of human

CNTF-R (amino acids 1–346) was linked via a single glycine

residue to the N-terminus of human CNTF (amino acids

1–186). Recombinant Hyper-CNTF protein was expressed

in COS-7 cells. Hyper-CNTF efficiently induced dose￾dependent STAT3 phosphorylation and proliferation of

BAF-3 cells stably transfected with gp130 and LIF-R

cDNAs. While on BAF3/gp130/LIF-R cells, Hyper-CNTF

and LIF exhibited similar biological responses, the activity

of Hyper-CNTF on pheochromocytoma cells (PC12 cells)

was quite distinct from that of LIF. In contrast to LIF,

Hyper-CNTF stimulated neurite outgrowth of PC12 cells in

a time- and dose-dependent manner correlating with the

ability to phosphorylate MAP kinases. These data indicate

that although LIF and Hyper-CNTF use the same

heterodimeric receptor complex of gp130 and LIFR, only

Hyper-CNTF induces neuronal differentiation. The thera￾peutic potential of Hyper-CNTF as a superagonistic

neurotrophin is discussed.

Keywords: cytokines; differentiation; rat; PC12 cells; signal

transduction.

Ciliary neurotrophic factor (CNTF) is a survival and

differentiation factor for a variety of neuronal and glial cells.

It has been proposed to act as a lesion factor preventing

motor neuron degeneration after injury [1] and exerting

myotrophic activity on denervated skeletal muscle [2].

CNTF belongs to the IL-6 type family of neuropoietic

cytokines that comprises interleukin-6 (IL-6), interleukin-11

(IL-11), leukemia inhibitory factor (LIF), oncostatin M,

cardiotrophin-1 (CT-1), and novel neurotrophin-1 (NNT￾1)/cardiotrophin-like cytokine (CLC) [3–7]. All IL-6 type

cytokines use a membrane spanning 130-kDa glycoprotein,

gp130, as a signal transducing receptor subunit. The

biological response to CNTF is elicited by formation of a

multimeric receptor complex [8]. CNTF first binds

to a specific glycosyl-phosphatidylinositol-anchored a unit,

CNTF receptor (CNTF-R), which is not involved in

signaling. This is followed by the recruitment of gp130

and LIF receptor (LIF-R) as signal transducing b units,

which in turn form a disulfide-linked heterodimer that

activates the JAK/STAT and the Ras/MAP kinase path￾ways [6,9]. IL-6, CNTF as well as IL-11 and presumably

CT-1 and NNT-1 act via specific membrane receptors which

together with their ligands associate with signal transducing

b subunits thereby initiating cytoplasmic signaling. Cells

that only express signal transducing but no ligand binding

subunits for these cytokines are refractory to stimulation.

An unusual feature of the IL-6 cytokine family is that the

soluble forms of the ligand binding receptor subunits

generated by one cell type in complex with their ligands can

directly stimulate the signal transducing receptor b subunits

on different cell types which lack ligand binding a subunits

[10]. This process has been named trans-signaling [11,12].

The soluble form of CNTF-R (sCNTF-R) can be

produced by limited proteolysis or by phospholipase

C-mediated cleavage [13]. Evidence for the importance of

soluble cytokine receptors in neuronal signaling, differenti￾ation and survival responses has accumulated (reviewed in

[14]).

Most recently, it was shown that the CNTF-R is also the

cellular receptor for an additional cytokine, cardiotrophin￾like cytokine (CLC) [15]. This fact explains the different

phenotype of CNTF–/– and CNTF-R–/– mice. Whereas

CNTF–/– mice show a mild phenotype [16] CNTF-R–/– mice

die shortly after birth [17].

Correspondence to P. Ma¨rz, Institute of Physiology,

University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland,

Fax: + 41 61 267 3559, Tel.: + 41 61 267 3553,

E-mail: [email protected]

Abbreviations: CNTF, ciliary neurotrophic factor; sCNTF-R, soluble

CNTF receptor; IL-6, interleukin-6; LIF, leukemia inhibitory factor;

CT-1, cardiotrophin-1; NNT-1, novel neurotrophin-1; CLC,

cardiotrophin-like cytokine; JAK, Janus kinase; STAT, signal

transducer and activator of transcription; MAPK, mitogen activated

protein kinase; DMEM, Dulbecco’s modified Eagle’s medium.

*Note: these authors contributed equally to this work.

(Received 6 February 2002, revised 25 April 2002, accepted 3May 2002)

Eur. J. Biochem. 269, 3023–3031 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02977.x