Transduction of static pressure signal to expression of human granurocytes macrophage colony stimulating factor mRNA in chinese hamster ovary cells

JOURNAL. OF BKSCIENCI! AND ESIOENNGINEERINF Vol. 96, No. I, 79-82. 2003

Transduction of Static Pressure Signal to Expression of Human Granurocytes Macrophage Colony Stimulating Factor mRNA in Chinese Hamster Ovary Cells HENG GONG,’

MUTSUMI

TAKAGI,‘*

AND

TOSHIOMI

YOSHIDA’

International Center for Biotechnology, Osaka University, 2-l Yamada-oka, Suita, Osaka 565-0871, Japan’ Received

17 September

2002iAccepted

17 March

2003

The expression level of human granurocytes macrophage colony stimulating factor (hGM-CSF) mRNA in Chinese hamster ovary (CHO) DRlOOOL4N cells increased by 24% with pressurization. Treatment of cells with chelerythrine chloride (10 nM, 15 min), an inhibitor of protein kinase C (PKC), did not suppress the pressure-induced (0.9 MPa) expression of LGM-CSF mRNA, while it decreased the LGM-CSF gene expression level at normal pressure (0.1 MPa). Treatment with U0126 (20 pM, 60 min), a specific inhibitor of extracellular signal-related kinase (ERK1/2), decreased the expression level of hGM-CSF mRNA at 0.1 MPa to 80% of that without U0126 treatment. Similarly, treatment with U0126 decreased the pressure-induced (0.9 MPa) expression level of hGM-CSF mRNA to 79% of the control expression level at 0.1 MPa without treatment. The amount of intracellular phosphorylated ERK1/2 increased with pressurization (0.9 MPa). These results suggest that the pressure-induced expression of hGM-CSF mRNA in CHO DRlOOOL4N cells depends not on PKC but on the ERK1/2 signaling cascade. [Key words: &&tic pressure,Chinese hamster ovary (CHO), protein kinase C, signal transduction, extracellular si,gnal-relatedkinase] Although static pressure is effective operational parameters in increasing the supply rate of dissolved oxygen in mammalian cell cultures, there are only a few reports that describe on the effect of static pressure on mammalian cells. (l-3). We have previously demonstrated that the conversion rate of glucose to lactate in human embryo lung (HEL) cells at 0.25 MPa is less than tlhat at 0.12 MPa, and the production rate of tissue plasminogen activator (tPA) increased by 16% as pressure increased (4) In mouse hybridoma cells (AFP27) cultured at various constant static pressures ranging between 0.1 and 0.9 MPa, the specific production rate of a monoclonal antibody increased from 4.5 to 5.6x lo-‘” mgicelllh in proportion to the pressure increase (5). In these studies, the partial pressures of oxygen and carbon dioxide were controlled to be constant independent of pressure in order to avoid the difference in pH and dissolved oxygen concentration at different pressures. The human granulocyte-macrophage stimulating factor (hGM-CSF) is an important hematopoietic growth factor (6-8) and important clinically (9, 10). We previously reported that pressurized cultivation of Chinese hamster ovary (CHO) DRlOOOL4N cells (11) at 0.9 MPa increases the amount of hGM-CSF secretion through upregulation of hGM-CSF mRNA expression (12). However, there are few reports on the transduction pathway of the static pressure signal in CHO cells. In this study, the transduction pathway of the static pressure * Corresponding author. phone: +81-(0)6-6879-7456

signal to the expression of hGM-CSF mRNA in CHO cells was investigated. PMA and chelerythrine chloride were obtained from Sigma (St. Louis, MO, USA). U0126 was purchased from Promega (Madison, WI, USA) and dissolved in dimethylsulfoxide. CHO DRlOOOL4N cells producing hGM-CSF, which gene was regulated by SV40 promotor (1 l), were cultivated employing Iscove’s modified Dulbecco’s medium (catalog no. 17633; Sigma) supplemented with 10% dialyzed fetal calf serum (lot. no. 1025398; Gibco, Grand Island, NY, USA), 0.1 mgll streptomycin (Gibco), 100 U/I penicillin (Gibco) and 1 PM methotrexate (catalog no. M8407; Sigma). T-flasks for cell culture (25 cm’; Corning, Tokyo) containing 5 ml of the medium were inoculated with cells (2.4 x 10’ cells) and incubated in a conventional CO1 incubator (37”C, 5% CO,) to near confluence. Cells were treated with the specified agents as described below, and then, further incubated in a pressurized incubator. The pressurized incubator system consisted of a stainlesssteel vessel (length, 120 mm; inner diameter, 95 mm), sealed using flanges fitted with valves, a pressure gauge, and a PVA filter. After the T-flasks were put into the stainlesssteel vessel and the gas in the vessel was replaced with a gas mixture of 5% CO, and 95% air, they were pressurized between 0.1 (1 atm) and 0.9 MPa using nitrogen gas. The culture temperature was maintained at 37°C using a water bath. The partial pressures of 0, and CO? in the T-flasks were, respectively, 0.02 1 and 0.005 MPa, independent of static pres-

e-mail: [email protected] fax: +81-(0)6-6879-7454 79

.I. HlO\(

sure, in order to avoid the difference in pH and dissolved oxygen concentration at different pressures. The vessel was not opened until the end of cultivation. Cell concentration was determined by the trypan-blue method. All culture experiments were performed more than twice to confirm result reproducibility. After the growth to near confluence at 0.1 MPa as described above, the cells were incubated with phorbol 12myristate 13-acetate (PMA, 100 nM), an activator of protein kinase C (PKC) (13), for 4 h at 0. I or 0.9 MPa and, then the hGM-CSF mRNA expression level was determined. After the growth to near confluence at 0.1 MPa and treatment of cells with chelerythrine chloride ( 10 nM), a specific inhibitor of PKC (14), for 15 min or I .4-diamino-2,3-dicyano1,4-bis[2-amino-phenylthio] butadiene (UOI 26, 20 PM), a specific inhibitor of extracellular signal-related kinase (ERK1/2) (15) which works by inhibiting the kinase activity of mitogen-activated protein (MAP) kinase kinase (MAPKK or MEKlI2) for 60 min, the cells were incubated for 4 h without the agents at 0.1 or 0.9 MPa and the hGMCSF mRNA expression level was determined. Total RNA from CHO DRlOOOL4N cells (5x 1O’cells) w-aseluted using 80 ~1 of Rnase-free water from the RNeasy minikit (Qiagen, Hilden, Germany). The concentration of total RNA was determined by measuring the absorbance at 260 nm. The eluate (2 ~1) was used for the reverse transcription reaction employing Omniscript Reverse Transcriptase (Qiagen) and an Oligo d(T) primer. This reverse transcription product was used for PCR employing HotStarTaq Master Mix (Qiagen) and biotinylated primers for hGMCSF mRNA. The amplified product was determined using a probe-coated plate (hGM-CSF mRNA XpressPack kit; Chemicon. Temecula, CA, USA) and a calorimetric

Control

Control

PMA

Chelerythrine

I. Illor

\I,

OligoDetect detection system (Chemicon) at an absorbance of 450 nM (A&. The ratio of the concentration of hGMCSF mRNA (Aqjo) to the concentration of total RNA (A,,,,) was calculated as the cell-specific level of hGM-CSF mRNA. CHO cells in T-flask (25 cm’) were cultured until 7& 80% confluence and then incubated for IS-24 h in a medium containing 0.5% serum in order to reduce the basal ERK phosphorylation level. After replacing the medium with fresh medium, the cells were further incubated at 0. I or 0.9 MPa for 4 h at 37°C. The cells were washed in icecold phosphate-buffered saline and lysed in 150 pl of Laemmli’s sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.01% bromophenol blue). The lysates were sonicated for lo-15 s, boiled for 10 min, and centrifuged (10,OOOxg) at 4°C for 5 min. Protein concentration of the lysate supernatant was determined using a Bio-Rad protein assay kit. The lysate supernatant containing 50 pg of protein was electrophoresed in 4-20% SDS-Tris glycine gels (catalog no. 211010; Daiichi Pure Chemicals, Tokyo) and resolved proteins were transferred to nitrocellulose membranes (Schleicher & Schuell. Keene, NH, USA). After blocking with 0.5% nonfat dried milk (Wako, Osaka), each membrane was probed with an anti-phospho-ERK112 antibady (l/1000) (New England Biolabs, Beverly, MA, USA) followed by incubation with HRP-conjugated secondary antibody (1 ilOO0) and visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences, Tokyo). The effects of PMA and chelerythrine chloride on the expression of hGM-CSF mRNA at 0.1 and 0.9 MPa were respectively studied. As shown in Fig. 1A and IB, the expression level of hGM-CSF mRNA with PMA (4.75kO.23

0.1 MPa

0.9 MPa

0.9 MPa

0.1 ivm

0.9 MPa

0.9 MPa Cheletythrine

FIG. I. Effects of PMA and chelerythrine on the expression of hGM-CSF mRNA in CHO cells. After the growth at 0. I MPa, cells were incubated together with PMA for 4 h at (A) 0.1 or (C) 0.9 MPa, or cells were treated with chelerythrine chloride for 15 min, then incubated for 4 h without the agents at (B) 0.1 or (D) 0.9 MPa, respectively, and then the hGM-CSF mRNA expression level was determined. Data represent the mean&SD. *, WO.05, compared with 0.1 MPa; #, P
NOTES

VOL. 96, 2003

0.1 MPa

0.1 MPa U0126

81

U0126

FIG. 2. Effect of UO126 on the expression of hGM-CSFmRNA in CHO cells.After the growth at 0.1 MPa, cells were treated with UOI 26 for 60 min and,then incubatedfor 4 h at (A) 0.1 and (B) 0.9 MPa.

in arbitrary units) was higher than that without PMA (3.05f0.33) (n=3, PcO.05). On the other hand, treatment with chelerythrine chlorilde (10 nM, 15 min) distinctly decreased the expression level of hGM-CSF mRNA to 78.8% of that without chelerythrine chloride treatment (n=3, P
0.1

0.1

0.9

Phospho-p44

(ERKl)

hospho-p42

(ERK2)

0.9

Pressure (MPa) FIG. 3. Effect of pressureon ERK phosphorylation.After CHO cells were incubated in a serum-deficient medium for 24 h, cells were subjected to pressures of 0.1 or 0.9 MPa for 4 h. Cellular protein was resolved by SDS-PAGE,and ERK phosphorylationwas assessedby

immunoblotting. promoters, we first tested whether or not hGM-CSF expression in CHO cells is PKC-dependent. The up-regulation by PMA (Fig. 1A) and downregulation by chelerythrine chloride (Fig. IS) of the hGM-CSF expression in CHO cells at 0.1 MPa clearly showed that hGM-CSF expression in CHO cells is partly PKC-dependent. Chelerythrine chloride did not suppress pressure-induced hGM-CSF mRNA expression (Fig. lD), although this inhibitor partially blocked hGM-CSF gene expression in CHO cells at normal pressure (0.1 MPa) (Fig. 1B). The percentage increase of the hGM-CSF mRNA expression level upon pressurization with PMA treatment was 76% (Fig. lC), which was almost equal to the sum of the percentage increase upon separate treatment with pressure (24%) (Fig. 1C) and PMA (56%) (Fig. 1A). These observations suggest that the expression of hGM-CSF mRNA in CHO cells in response to high pressure is PKC-independent. There must be another signaling pathway that transduces a pressure-signal for the higher expression level of hGM-CSF mRNA in CHO DRlOOOL4N cells. Treatment with U0126 decreased the hGM-CSF mRNA expression level to 79% of that without U0126 treatment at normal pressure (Fig. 2A). The pressure-induced hGM-CSF mRNA expression was also suppressed by treatment with U0126, which resulted in an expression level lower than that at normal pressure, 80% of that without U0126 treat-

ment at 0.1 MPa (Fig. 2B). This indicates that the inhibition of ERK 112 completely blocks the pressure-induced expression of hGM-CSF mRNA. Moreover, pressurization of CHO cells at 0.9 MPa resulted in stronger phosphorylation of ERKl/2 than that at 0.1 MPa (Fig. 3) which indicates that ERKl/2 phosphorylation in CHO cells is induced by high pressure. Although the expression level of ERKII2 may be affected by pressure, these results suggest that pressure affects the hGM-CSF gene expression in CHO DRlOOOL4N cells via the ERK112 signaling cascade. Although the mechanism that ERK activation leads the activation of SV40 promoter may contain the activation of an enhancer, this issue should be studied in future. In conclusion, the pressure-induced gene expression of hGM-CSF is PKC-independent and is almost completely via the ERK l/2 signaling cascade in CHO DRI OOOL4N cells, while the basal expression of hGM-CSF at normal pressure (0.1 MPa) is accompanied by the activation of PKC and ERK I /2 in CHO cells. Generality of such an effect of static pressure should be studied in future for other proteins and cells.

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