Protein kinase ERK contributes to differential responsiveness of human myeloma cell lines to IFNα
© Song et al; licensee BioMed Central Ltd. 2002
Received: 18 June 2002
Accepted: 8 July 2002
Published: 8 July 2002
Despite IFNα has been used extensively in the treatment of multiple myeloma (MM), there are also several reports suggesting that IFNα may aggravate isease in some MM patients. That means the effect of IFNα on the growth of myeloma cells in vivo may be different. In this study, we selected two human myeloma cell lines that vary remarkably in response to IFNα and focused on elucidating the mechanism of differential IFNα responsiveness.
Sko-007 is a myeloma cell line whose growth is arrested by IFNα; however, IFNα promoted the proliferation of the other myeloma cell line U266. We observed that the growth-stimulation effect of IFNα on U266 cells did not result from up-regulation of the IL-6 receptors on cell surface; while IFNα treatment on Sko-007 cells significantly reduced gp130 expression. Moreover, the transcription factors STAT3 and STAT1, which are involved in the JAK/STAT signal transduction pathway, can be activated in both IFNα-stimulated and -inhibited myeloma cell lines; while the activation of the protein kinase ERK, which is involved in the Ras/MAPK signal transduction pathway, can be down-regulated in IFNα-arrested Sko-007 cells and up-regulated in IFNα-stimulated U266 cells. In addition, both IFNα-induced growth-stimulation effect and the up-regulated activation of ERK in U266 cells were efficiently inhibited by PD98059, the specific inhibitor of MAPK/ERK kinase (MEK).
Myeloma cells responsiveness to IFNα is heterogeneous and the activation state of ERK in the Ras/MAPK signalling pathway mainly contributed to this difference.
The typically growth-inhibitory action of Interferonα (IFNα) has made it a commonly used therapeutic agent in the treatment of a wide variety of human malignancies, including multiple myeloma (MM) . However, although a considerable number of the clinical trials have addressed the effectiveness of IFNα on MM therapy , other reports also showed that in some MM patients, IFNα can promote the proliferation of myeloma cells in vivo and result in the development of aggressive plasma cell leukemia (PCL) . Therefore, the action of IFNα on MM cell growth is a matter of debate, and the mechanism of this discrepancy is the topic of this study.
Interleukin-6 (IL-6) is the major growth and survival factor for myeloma cells. IFNα and IL-6 clearly use distinct receptor complexes; however, both cytokines have been shown to use two common signal transduction pathways to mediate their biological activities. One is the janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway which results to the activation of the protein tyrosine kinases JAKs and the transcription factors STAT3 and STAT1; the other is the Ras-dependent mitogen-activated protein kinase (Ras/MAPK) pathway which involves the subsequent activation of a series of serine/threonine kinases including extracucullar signal-regulated kianse (ERK) [4, 5]. In the previous report, we have investigated in detail the mechanism of IL-6 signal transduction in different human myeloma cell lines . In this study, we analyzed IFNα-induced activation of STAT3, STAT1 and ERK in Sko-007 and U266 human myeloma cell lines, which displayed overall different response to IFNα. And we demonstrated the evidence that the activation state of the Ras/MAPK in stead of the JAK/STAT signal transduction pathway accounted mainly for the differential effect of IFNα on myeloma cells.
Myeloma cell lines are heterogeneous in response to IFNα
Effect of IFNα on the expression of two IL-6 receptor chains on myeloma cell surface
Expression of IL-6R and gp130 on the surface of Sko-007 and U266 cells in the absence or presence of IFNα.
13.00 ± 1.21
20.57 ± 2.74
10.11 ± 0.96
19.80 ± 0.47
99.06 ± 1.01
60.68 ± 1.70
65.77 ± 3.19*
34.21 ± 1.31*
1.50 ± 0.33
50.44 ± 2.09
0.92 ± 0.36
50.09 ± 4.28
8.66 ± 0.64
29.19 ± 2.13
9.33 ± 1.05
26.66 ± 0.63
Activation of the transcription factors STAT3 and STAT1 in myeloma cells stimulated by IFNα
Activation state of the protein kinase ERK in myeloma cells stimulated by IFNα
PD98059 inhibited IFNα-induced growth-stimulation effect and up-regulation of ERK activation in U266 cells
Up till now, the clinical value of IFNα on multiple myeloma therapy remains debatable due to the conflicting in vivo results reported by different clinical groups [2, 3]. Therefore in this study, we analyzed the responsiveness of a panel of human myeloma cell lines to IFNα in vitro and the mechanism of myeloma cells heterogeneous response to IFNα.
The results in this study showed that IFNα exerted different effects on the growth of different myeloma cell lines. The growth-inhibition effect of IFNα was significant while the growth-stimulation effect was moderate (Fig. 1). This result is in accordance with the fact that IFNα usually exerts a strongly inhibitory or a slowly stimulatory effect on the proliferation of myeloma cells in different patients with MM [2, 3].
To elucidate the mechanism responsible for the different activity of IFNα on myeloma cells, we firstly examined the regulation of IL-6R and gp130 expression on cell surface in the presence of IFNα, which may be one of the reasons for the heterogeneous IFNα responsiveness according to the previous reports . Interestingly, we only detected down-regulated expression of gp130 in IFNα-arrested Sko-007 cells, while not any changes on two IL-6 receptor chains level in IFNα-stimulated U266 cells. Therefore, the regulation of IL-6 receptors expression may not mainly account for the different effect of IFNα on myeloma cells.
In the further study, we focused on investigating the activation states of IFNα-induced signal transduction pathways in myeloma cellls. We observed that both STAT3 and STAT1 could be activated in the both two myeloma cell lines irrespective of growth outcome upon IFNα stimulation; while the activation state of ERK correlated tightly with the growth effect of IFNα (Fig. 2, 3 and 4). That means, the Ras/MAPK in stead of the JAK/STAT signal transduction pathway mainly contributed to the different response of myeloma cells to IFNα. Identification of the target gene(s) regulated by protein kinase ERK in the Ras/MAPK pathway in IFNα-arrested and stimulated myeloma cells will be helpful to further elucidate the mechanism of IFNα on myeloma cells.
Our data in this study clearly demonstrated that IFNα can exert growth-stimulatory or -inhibitory effect on different myeloma cells in vitro and protein kinase ERK played a key role in myeloma cells in response to IFNα.
Materials and methods
Human recombinant IFNα was provided by Institute of Biotechnology (Beijing, China). Anti-human IL-6R and gp130 antibodies were prepared in our lab. Anti-ERK and phospho-ERK antibodies was purchased from Santa Cruz Biotechnology (USA). Anti-STAT3, STAT1, phospho-STAT3 and phospho-STAT1 antibodies were purchased from NEB Biotechnology (USA).
Human myeloma cell lines
Human myeloma cell line U266 was kindly provided by Prof. Xueguang Zhang (Suzhou University, China). Sko-007 myeloma cell line was obtained from Medical School of Stanford University (USA). The cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin and 80 U/ml streptomycin.
Effect of IFNα on the growth of myeloma cells
The proliferation of myeloma cells in the presence of IFNα was measured by MTT assay as described previously .
Expression of two IL-6 receptor chains (IL-6R and gp130) on myeloma cell surface
2 × 105 myeloma cells were treated with or without IFNα (100 U/ml) for 48 h and then incubated with anti-IL-6R or anti-gp130 antibodies for 30 min and with the relevant FITC-labeled second antibodies for 20 min. Cell samples were harvested and subjected to FACS analysis.
Activation of the protein kinase ERK and the transcription factors STAT3 and STAT1 in myeloma cells induced by IFNα
2 × 106 myeloma cells were treated with IFNα (100 U/ml) for 10 min (to detect the activation of STAT3 and STAT1) or 48 h (to detect the activation of ERK) and then lyzed in 100 μL lysis buffer (10 mM Tris-Cl pH 7.6, 150 mM NaCl, 0.5% NP40, 5 mM EDTA, 1 mM PMSF, 2 mM Na3VO4, 1 mM NaF, 2 μg/mL aprotinin). After 30 min on ice, cell lysates were cleared by configuration at 12000 × g for 30 min at 4°. The concentrations of the cytoplasmic proteins were measured by Bradford assay . Then 30 μg cytoplasmic proteins were separated by 10% polyacrylamide-SDS gel and transferred elechtrophoretically to Immobilon (Millipore). After blocking with 5% dry milk, the membranes were blotted with anti-ERK, STAT3, STAT1 or anti-phospho-ERK, phospho-STAT3, phospho-STAT1 antibodies and the relevant HRP-labeled second antibodies. Immunoreactive proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham, USA).
We thank Professor Xueguang Zhang (Institute of Biotechnology, Suzhou University, China) for providing U266 myeloma cell line. This project is supported by the National Natural Science Foundation of P. R. China (No.39925019).
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