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. 2008 Jan;10(1):52–60. doi: 10.1593/neo.07691

Oncogenic Ras-Induced Morphologic Change Is through MEK/ERK Signaling Pathway to Downregulate Stat3 at a Posttranslational Level in NIH3T3 Cells1

Hsuan-Heng Yeh *,†,‡, Chin-Han Wu *, Raghavaraju Giri †, Ken Kato §, Kimitoshi Kohno §, Hiroto Izumi §, Cheng-Yang Chou ‡,‖, Wu-Chou Su *,¶,#, Hsiao-Sheng Liu *,†,#
PMCID: PMC2213899  PMID: 18231638

Abstract

Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3) can be activated by cytokine stimulation. In this study, we unravel that Ha-rasV12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-rasV12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-rasV12 was through mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) signaling pathway. Microtubule disruption is involved in Ha-rasV12-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-rasV12-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

Introduction

Ha-, K-, and N-Ras are small G proteins which act as molecular switches to control signaling pathways that are key regulators of normal cell growth and malignant transformation [1]. The signaling pathways downstream of Ras include the Raf/mitogen-activated protein kinase (MAPK) pathway [2], phosphatidylinositol 3-kinase (PI3K)/Akt pathway [3], and phospholipase C/protein kinase C pathway [4]. Ras-transformed fibroblast cells undergo drastic morphologic change probably by decreased focal adhesion or disruption of the cellular cytoskeleton. Ha-ras overexpression disrupts microtubule formation through ERK activation and has been previously reported [5]. The alteration of actin filaments in Ras-transformed cells also correlates with decreased expression of various cytoskeletal proteins, such as α-actinin [6], vinculin [7], or the high-molecular weight tropomyosin isoforms [8,9].

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that become activated in response to growth factors and cytokines. Upon activation, activated STATs form homo- or heterodimers and are translocated into the nucleus, bind to the promoter region of specific target genes, and regulate gene transcription [10]. STAT proteins play an important role in signaling pathways critical to a wide variety of biologic processes, including cell proliferation, survival, carcinogenesis, differentiation, and development [11,12].

Cell movement normally accompanies morphologic change. The role of STATs in cell motility has been reported. Briefly, in Drosophila ovary, STAT is required for the migration of the border cells [13]. In zebrafish embryos, signal transducer and activator of transcription 3 (Stat3) is also essential for migration of sheets of cells during gastrulation stage [14]. In addition, keratinocytes of Stat3 knockout mouse exhibit migration defects of wounding ability [15]. Activated Stat3 is a component of focal adhesion within ovarian cancer cells and mouse fibroblasts [16]. Together, the above findings raise the possibility that activated Stat3 may contribute to cell motility by responding to changes in cell adhesion or by affecting the cytoskeleton. Recently, it has been reported that Stat3 is required for the stabilization of microtubules and cell migration [17]. In summary, Stat3 seems essential for a morphologic change during cell migration and transformation.

In this study, we unravel that Ha-rasV12 overexpression-induced NIH3T3 morphologic change of cells is through downregulation of Stat3 protein at a posttranslational level.

Materials and Methods

Materials

The active-form Stat3 (pRcCMV-Stat3C) was kindly provided by Dr. James Darnell, Jr. [18]. The reporter plasmid of Stat3 promoter (pST3LUC-2) was described previously [19]. Pharmaceutical inhibitors PD98059, U0126, LY294002, SB203580, SP600125, and rapamycin were obtained from Biomol International L.P. (Plymouth Meeting, PA). MG132 was obtained from Calbiochem (San Diego, CA). Ras antibody was obtained from Oncogene Science (San Diego, CA). Stat3 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). β-Actin antibody was obtained from Sigma-Aldrich (St Louis, MO). The antibodies for p70S6K1-Thr389, ERK-Thr202/Tyr204, ERK, Akt-Ser473, Akt, mTOR-Ser2481, and Stat3-Tyr705 were obtained from New England Biolabs (Beverly, MA). Monoclonal anti-acetylated β-tubulin (6-11B-1) was purchased from Sigma Chemical (St. Louis, MO). Fluorescein-conjugated goat anti-mouse secondary antibody was purchased from Chemicon (Hofheim, Germany). Isopropyl β-d-thiogalactopyranoside (IPTG) was purchased from Invitrogen (Boston, MA).

Cell Lines

Mouse fibroblast NIH3T3 cells harboring the inducible Ha-rasV12 oncogene (pSVlacOras) cloned from T24 cell line was designated as 7-4 [20]. The 7-4 derivates, 7-4-ST3 cells, contain active-form Stat3 (pRcCMV-Stat3C). All the cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, GIBCO, Gaithersburg, MD) supplemented with 10% calf serum (GIBCO) at 37°C in a 5% CO2 incubator.

Cell Lysis

The cell lysates were prepared as described before [21]. Briefly, the cells were washed twice with ice-cold PBS and lysed with 200 µl per 10-cm plate of whole-cell extract lysis buffer (50 mM Tris, pH 7.4, 1% NP-40, 2 mM EDTA, 100 mM NaCl, 10 mM Na orthovanadate, 0.1% SDS, 10 mg/ml leupeptin, 2 mg/ml aprotinin, and 100 mM phenylmethylsulfonyl fluoride) (protease inhibitors from Roche Applied Sciences, Indianapolis, IN). Cell lysates were further cleaned using centrifugation at 14,000 rpm for 10 minutes. Protein concentration was determined using a Bradford assay (Bio-Rad, Richmond, CA).

Western Blot Analysis

Equal amounts of cell lysates were boiled for 5 minutes with the sample buffer before separation on an SDS-polyacrylamide gel. The proteins in the gel were transferred onto a nitrocellulose filter (Millipore, Billerica, MA) in Tris-glycine buffer at 100 V for 1.5 hours using an electroblotter (Amersham Biosciences Corp., Piscataway, NJ). Membranes were blocked with PBS buffer containing 5% nonfat milk before incubating with antibodies. The target protein binding the specific antibody was detected by enhanced chemiluminescence according to the manufacturer's recommendations (Amersham).

RNA Extraction and Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted using a single-step method with TRIzol reagent (Invitrogen Corp., Carlsbad, CA). For reverse transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized from 0.2 to 1 µg of total RNA with an oligo-dT primer and the Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI). The sequences of PCR primers were as follows: Stat3 sense primer, 5′-GATGCGACCAACATCCTGGTG-3′; Stat3 antisense primer, 5′-GGACATCGGCAGGTCAATGGT-3′; β-actin sense primer, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′; and β-actin antisense primer, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′. The PCR protocol was conducted with the Stat3 primers at 94°C for 30 seconds, 57°C for 1 minute, and 72°C for 1 minute (30 cycles), followed by 72°C for 10 minutes. The PCR protocol was performed with the β-actin primer at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute (25 cycles), followed by 72°C for 10 minutes.

Luciferase Reporter Assays

Samples of 1 x 105 cells in 35-mm plates in triplicate were transfected with desired plasmid DNA using a cationic liposome (Lipofectin; GIBCO BRL, Life Technologies, Inc., Grand Island, NY). The plasmids of pST3LUC-2 Stat3 reporter gene (0.5 µg) and β-galactosidase (β-gal) expression vector (0.1 µg) were cotransfected into the cells. The medium was replaced with fresh DMEM 16 hours after transfection. Cells were then treated with 5 mM IPTG for another 24 hours, and then harvested. Luciferase and β-gal activities of the cell extracts were then determined, using the luciferase assay system and β-galactosidase enzyme assay system (Promega). Luciferase activity was then normalized using β-gal activities as the internal control.

Immunofluorescence Assay

7-4 and 7-4-ST3 cells were grown on glass coverslips overnight. After IPTG treatments, cells were washed with PBS, fixed in 50% acetone/methanol in cold PBS, and blocked with blocking reagent (DakoCytomation, Inc., Carpinteria, CA) for 1 hour to block nonspecific binding sites. Cells were then incubated with anti-acetylated α-tubulin antibody diluted 1:500 in blocking reagent for 1 hour. Cells were washed with PBS and incubated with fluorescein-conjugated goat anti-mouse secondary antibody at a 1:100 dilution. The cells were then mounted and the immunofluorescent images were taken by a confocal microscope (FV-1000; Olympus, Tokyo, Japan).

Results

Oncogenic Ha-ras Overexpression Suppresses Stat3 Expression through MEK/ERK Pathway

In this study, the 7-4 cells derived from NIH3T3 cells containing an inducible Ha-ras oncogene under the regulation of Lac system was used [22]. Briefly, bacterial lactose repressor system (Lac system) can regulate the expression of a reporter gene by the binding of lac repressor to a lac operator sequence, which is located between the TATA box and the transcription staring site of the gene in mammalian cells and mice. The reporter gene can be specifically activated by the addition of the lactose analogue, IPTG, which binds to the repressor and disables it from binding to the operator sequence by conformational change. Initially, the Stat3 protein expression levels in 7-4 cells in the presence or absence of IPTG were evaluated. Figure 1 shows that Ras induction, compared with the control of without IPTG, gradually suppressed Stat3 expression while the time of induction was increased. The suppressive effect of Ras on Stat3 expression is also demonstrated in a Ras-dependent fashion (Figure 2, the panels for Stat3 and Ras). In conclusion, an evident correlation between Ras overexpression and Stat3 downregulation was demonstrated in the stable NIH3T3 cell line 7-4.

Figure 1.

Figure 1

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Ha-rasV12 overexpression downregulates protein expression level of Stat3. The 7-4 cells were seeded in 10-cm dishes for 24 hours, and then treated with or without IPTG (5 mM) for 3, 6, 9, 12, 18, and 24 hours, respectively. Cells were harvested and the lysates were subjected to Western blot analysis to detect Stat3, Ras, and β-actin using specific antibodies against each protein.

Figure 2.

Figure 2

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Ha-rasV12 overexpression activates JNK, ERK, and PI3K pathways. The 7-4 cells that were seeded in 10-cm dishes for 24 hours were treated with different dosages of IPTG as indicated for another 24 hours. Cell lysates harvested were subjected to Western blot analysis to detect the expression levels of various proteins using specific antibodies as indicated. The band's phosphorylation intensity of JNK, ERK, and Akt was quantified by densitometry.

We then clarified the signaling pathways downstream of Ras involved in the suppression of Stat3 under Ras-overexpressed conditions. Consistent with other reports, phosphorylation of extracellular-regulated kinase (ERK), Jun N-terminal kinase (JNK), p38, and Akt were increased by Ras overexpression in 7-4 cells (Figure 2). Quantified results show that phosphorylation of ERK is impressive and changes in phosphorylation of JNK and Akt are subtle. In contrast, the total protein levels of ERK and Akt were not affected (Figure 2). Pharmaceutical inhibitors of specific signaling pathways were used to further clarify the signaling pathways downstream of Ras that participated in the suppression of Stat3. Treatment of the Ras-overexpressed cells with MEK/ERK pathway-specific inhibitors, PD98059 or U0126, the ERK phosphorylation induced by Ras was inhibited in a dose-dependent manner (Figure 3, A and B, pERK panel). Those inhibitors do not inhibit Ras-induced Akt phosphorylation (Figure 3, A and B, pAkt panel). The level of Stat3 protein expression suppressed by Ras was gradually restored, whereas the Ras-overexpressed cells were treated with increasing dosages of PD98059 or U0126 (Figure 3, A and B, Stat3 panel), indicating that MEK/ERK pathway is involved in Ras-related suppression of Stat3 expression. Treating the same cells with the PI3K inhibitor, LY294002, the Ras-induced Akt phosphorylation was inhibited dose-dependently (Figure 3C, pAkt panel), but the levels of ERK phosphorylation induction was not affected by LY294002 (Figure 3C, pERK panel). Moreover, the suppression of Stat3 expression by Ras could not be restored (Figure 3C, Stat3 panel). The p38 and JNK pathway inhibitors, SB203580 and SP600125, could not reverse the Ras-related suppression of Stat3 expression (data not shown). Taken together, the above results clearly demonstrate that downregulation of Stat3 by oncogenic Ha-ras is mainly through activation of MEK/ERK and not PI3K/Akt, p38, or JNK signaling pathways.

Figure 3.

Figure 3

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Ha-rasV12 overexpression downregulates Stat3 expression through MEK/ERK pathway. The 7-4 cells were seeded in 10-cm dishes for 24 hours and then treated with IPTG (5 mM) in the presence or absence of the pharmaceutical inhibitors (A) PD98059, (B) U0126, or (C) LY294002 for another 24 hours. The cells were harvested and the lysates were subjected to Western blot analysis to detect various protein expression levels by using specific antibodies as indicated.

mTOR/p70S6K Pathway-Related Translational Control Does Not Participate in Stat3 Expression By Oncogenic Ha-ras Overexpression

To determine at what level that the Stat3 expression was downregulated by Ras, we analyzed the effects of Ras overexpression on RNA expression of Stat3 as well as on the Stat3 promoter activity. We found that the mRNA level of Stat3 was unchanged whether or not Ras was overexpressed (Figure 4A). Similarly, Ras overexpression does not affect the activity of the Stat3 promoter reporter gene (Figure 4B). Altogether, our data indicate that downregulation of Stat3 protein expression by Ras is not at the transcriptional level. We then clarified the possibility that downregulation of Stat3 protein expression by Ras is through mTOR/p70S6K signaling pathway. mTOR, one of the Ras-downstream molecules, is known to participate in the translational control of protein expression. The activation of mTOR/p70S6K pathway in Ras-overexpressed 7-4 cells was investigated. Figure 4C shows that the levels of mTOR and its downstream p70S6K were dose-dependently increased by oncogenic Ha-ras. To further study whether the translational control of the suppression of Stat3 expression by Ha-ras oncogene is mediated through mTOR/p70S6K pathway activation, rapamycin was used to inhibit Ras-induced mTOR activation. Figure 4D shows that 7-4 cells treated with rapamycin inhibited Ras overexpression-induced phosphorylation of mTOR and p70S6K (pmTOR and pP70S6K panels). However, the levels of Stat3 protein expression downregulated by Ras overexpression cannot be reversed (Stat3 panel), indicating that mTOR/p70S6K pathway-related translational control of protein expression is not involved in suppression of Stat3 expression by Ras.

Figure 4.

Figure 4

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Stat3 expression is downregulated by Ha-rasV12 through an mTOR/p70S6K-independent pathway. (A) The 7-4 cells, after seeding for 24 hours, were treated with IPTG (5 mM) for another 24 hours. Cells were harvested and RNA was isolated for RT-PCR analysis of Stat3 expression. β-Actin was used as the internal control. (B) The Stat3 luciferase reporter gene was transiently transfected into the cells. Twenty-four hours after transfection, the cells were treated with IPTG (5 mM) for another 24 hours. The cells were then harvested and the cell lysates were subjected to analysis of luciferase activities using the luciferase assay system. (C) The 7-4 cells were treated with various concentrations of IPTG for 24 hours. (D) The 7-4 cells, after seeding for 24 hours, were treated with IPTG (5 mM) or combined with different dosages of rapamycin for another 24 hours. Western blot analysis was conducted to detect the expression levels of Stat3, phosphor-p70S6K, phosphor-mTOR, and Ras using the specific antibodies as indicated.

Proteosome Degradation Is Involved in Downregulation of Stat3 By the Oncogenic Ha-ras

Our data indicate that downregulation of Stat3 protein expression by Ras is not at transcriptional level and mTOR/p70S6K signaling pathway is also not involved. Figure 5A shows that protein half life of Stat3 in 7-4 cells is 21 hours after IPTG treatment.We then clarified the possibility that downregulation of Stat3 protein expression by Ras is through proteosome degradation. The 26S proteosome inhibitor, MG132, was used to inhibit proteosome degradation of Stat3. Figure 5B shows that Stat3 protein expression downregulated by Ras overexpression could be reversed by treatment of the Ras-overexpressed cells with MG132 in a dose-dependent manner. These data clarify that downregulation of Stat3 protein expression by Ha-ras oncogene was through 26S proteosome-dependent protein degradation.

Figure 5.

Figure 5

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Proteosome degradation is involved in the downregulation of Stat3 by Ha-rasV12 overexpression. (A) The 7-4 cells seeded in 10-cm dishes for 24 hours were treated with or without 5 mM IPTG for 3, 6, 9, 12, 18, and 24 hours, respectively. Cells were harvested and the lysates were subjected to Western blot analysis to detect Stat3. The band's intensity was quantified by densitometry. (B) The 7-4 cells, after IPTG (5 mM) treatment for 24 hours, were then treated with or without MG132 (20µM) for 1, 3, and 5 hours, respectively. Harvested cell lysates were subjected to Western blot analysis and the expression levels of Stat3, Ras, and β-actin were detected using Stat3, Ras, and β-actin antibodies, respectively.

Overexpression of Oncogenic Ha-ras-Induced Morphologic Change Is Reversed By the Inhibitors of MEK/ERK Pathway

Altered cellular morphology is one of the characteristics of cell transformation [23]. Ras overexpression could induce morphologic change of mouse NIH3T3 fibroblasts, normal rat kidney (NRK) cells, as well as human MSU-1.1 cells [24]. Similarly, the morphology of 7-4 cells was altered from flat-like (Figure 6A) to round-up-like (Figure 6B), whereas Ras was overexpressed. Because MEK/ERK, and not other signaling pathways, was involved in Ha-ras oncogene-related downregulation of Stat3, we checked whether the morphologic change induced by oncogenic Ha-ras could also be reversed by inhibition of the MEK/ERK signaling pathway. As expected, the cells treated with either PD98059 or U0126 caused apparent reversion of morphology from round to flat (Figure 6, C and D). In contrast, SB203580, SP600125, LY294002, or rapamycin caused no reversion of morphology (Figure 6, E–H). Taken together, Ras overexpression-induced morphologic change is dependent on MEK/ERK pathway.

Figure 6.

Figure 6

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Ha-rasV12 overexpression-induced morphologic change is blocked by the inhibitor of MEK/ERK pathway. The 7-4 cells were seeded in the plate and were (A) left untreated or were treated with (B) IPTG (5 mM) alone or in combination with (C) PD98059, (D) U0126, (E) SB203580, (F) SP600125, (G) LY294002, or (H) rapamycin for 24 hours. The pictures of cellular morphology were taken under the light microscope at a magnification of x100.

Oncogenic Ha-ras-Induced Morphologic Change Is Reversed By Increasing Stat3 Expression

Because oncogenic Ha-ras-induced morphologic change accompanies downregulation of Stat3, the relationship between morphologic change and Stat3 downregulation was then investigated. A 7-4 derivate, 7-4-ST3 cell line overexpressing active-form Stat3, was established to increase Stat3 protein expression regardless whether Ras was overexpressed or not (Figure 7A, Stat3 panel and Ras panel). The morphology of 7-4-ST3 cells harboring the constitutively expressed Stat3 becomes much flatter compared to that of 7-4 cells, whereas Ras was overexpressed in the presence of IPTG (Figure 7B). These findings sustain the notion that Stat3 protein is crucial for Ras-mediated morphologic change in 7-4 cells. To clarify whether overexpression of Stat3 affects the signaling pathways activated by oncogenic Ha-ras, the activities of the major signaling pathways MAPK and PI3K/Akt downstream of Ras were evaluated. Figure 7A shows that activation of MAPK/ERK and PI3K/Akt signaling pathways by Ras was not affected by increasing the expression of Stat3 in 7-4-ST3 cells. Our data clearly demonstrate that Ras-induced morphologic change is associated with Stat3 expression, and that the reverse of Ras-induced morphologic change is not through MAPK/ERK and PI3K/Akt signaling pathways.

Figure 7.

Figure 7

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Ha-rasV12 overexpression-induced morphologic change can be reversed by the restoration of Stat3 expression. (A) The 7-4 and 7-4-ST3 cells were seeded in 10-cm dishes for 24 hours, and then treated with 0, 0.5, or 5 mM IPTG for 24 hours. The cells were harvested and the cell lysates were subjected to Western blot analysis and detected using various antibodies indicated. (B) The 7-4 and 7-4-ST3 cells were seeded in 10-cm dishes for 24 hours, and then treated with IPTG (5 mM) for another 24 hours. The pictures of cellular morphology were taken under the light microscope at the magnifications of x100 and x200, respectively.

Oncogenic Ha-ras-Induced Morphologic Change Is Due to the Disruption of Microtubules and Stat3 Stabilizes Microtubules

It has been reported that Ha-ras overexpression disrupts microtubule formation through ERK activation [5]. Stat3 can stabilize microtubule through interaction with stathmin [17]. Therefore, it is intriguing to know whether microtubules are involved in Ras- and Stat3-related morphologic change. Figure 8 shows that acetylated microtubule networks were disrupted when Ras was overexpressed in 7-4 cells (Figure 8, E vs F). Conversely, acetylated microtubule networks of 7-4-ST3 cells remained intact although Ras was overexpressed (Figure 8, G vs H), suggesting that Ras overexpression-induced morphologic change is due to the disruption of microtubules. In contrast, Stat3 stabilizes microtubules, therefore causing the reversion of Rasinduced morphologic change.

Figure 8.

Figure 8

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Stat3 overexpression restores Ha-rasV12 overexpression-induced microtubule disruption. The 7-4 and 7-4-ST3 cells were seeded onto cover slides for 24 hours, and then treated with IPTG (5 mM) for another 24 hours. The pictures of cellular morphology were taken under the light microscope at a magnification of x400 (upper panel, A to D). For microtubule staining, 7-4 and 7-4-ST3 cells treated with or without IPTG were assayed by immunofluorescence with anti-acetylated α-tubulin antibody (lower panel, E to H). Images were collected by a confocal microscope.

Discussion

We found that Ha-rasV12 overexpression in NIH3T3 cells could induce cell morphologic change through downregulation of Stat3, which is mediated through the MEK/ERK signaling pathway. The suppression of Stat3 expression by Ras is not at the transcriptional level but through proteosome degradation. We demonstrate that increasing the expression level of Stat3 reverses Ha-rasV12 overexpression-induced morphologic change.

Altered cellular structure, shape, and cytoskeletal organization are characteristics of cellular transformation [23]. Cytoskeletal change in tumor cells is crucial for transformation [25]. The mechanisms of Ras-mediated transformation involve a number of downstream signaling pathways and activation of Raf/MEK/ERK is sufficient to transform NIH3T3 fibroblasts [26,27]. We show that PD98059 or U0126 treatment effectively blocked the morphologic change of Ha-rasV12-overexpressed 7-4 cells (Figure 6). In addition, increasing Stat3 expression reversed the morphologic change of the Ha-rasV12-overexpressed 7-4 cells (Figure 7B). Therefore, in Ha-rasV12-transformed mouse NIH3T3 fibroblast, the morphologic change induced by Ha-rasV12 is dependent onMEK/ERK-induced suppression of Stat3 expression.

Ras overexpression-induced morphologic change occurred not only in H-ras-transformed mouse NIH3T3 fibroblast but also in K-ras-transformed rat NRK cells and human MSU-1.1 fibroblast cells [24]. In NRK rat fibroblast cells, treating the cells with U0126 could reverse K-ras-mediated morphologic change by blocking both MEK/ERK and p70S6K pathways [28]. In the Ha-rasV12-overexpressed NIH3T3 mouse fibroblast, although rapamycin could inhibit Ha-rasV12 overexpression-induced p70S6K activation (Figure 4D), the morphologic change induced by Ha-rasV12 could not be blocked (Figure 6H), suggesting that Ha-rasV12 overexpression-induced morphologic change is mTOR/p70S6K-independent in mouse NIH3T3 cells. Taken together, Ras overexpression-induced morphologic change depends on different signaling pathways that are cell type-specific.

Increased adhesiveness and fast spreading on the collagen matrix was found in Stat3-deficient keratinocytes, and deficiency of Stat3 in the cells leads to the hyperphosphorylation of p130cas [29]. Because focal adhesion complex proteins, including p130cas, modulate multiple cell functions, including migration, proliferation, survival, and morphologic change, these results support our findings that the expression levels of Stat3 protein are critical for Ha-rasV12 overexpression-induced cell roundup. Stat3 has been shown to localize in focal adhesions and function in motility of ovarian cancer cells [16], suggesting the effects of Stat3 on focal adhesion complex proteins. According to this study, focal adhesion proteins other than p130cas, which may participate in Stat3 downregulation-induced morphologic change, are worthy of further investigation.

In NRK cells, farnesyltransferase inhibitor treatment to inhibit Ras activity can reverse Ras-induced morphologic change [30]. Suzuki et al. [30] further demonstrated that blockage of Ras activity affected microtubule dynamics that is involved in these processes. Lovric et al. [31] also showed that Raf activation-induced reorganization of the microtubule network is through the hyperphosphorylation of stathmin. Recently, Ng et al. [17] found that the expression of Stat3 is required for the stabilization of microtubules and cell migration through directly interacting with stathmin. Our data also showed that stabilization of microtubules in the 7-4 cells is disrupted by Ha-rasV12 overexpression. Moreover, increasing Stat3 expression in 7-4-ST3 cells could restore the microtubules' stability and morphologic change induced by Ras. Altogether, downregulation of Stat3 may affect the interaction of Stat3 with stathmin in 7-4 cells and further lead to destabilizing the microtubules. However, whether MEK/ERK pathway is involved in the interaction between Stat3 and stathmin in Ha-rasV12-overexpressed 7-4 cells needs to be further clarified.

Stat3 protein modification including ubiquitination has been reported. In H4IIE rat hepatoma cells, osmolarity could regulate Stat3 protein stability. The hyperosmolarity-accelerated Stat3 degradation could be prevented by proteosome inhibitors because of a decrease in Stat3 ubiquitination [32]. Selective degradation of Stat3 by the proteasome was also contributed to growth suppression and apoptosis of ubiquitin-treated hematopoietic cells [33]. In our studies, proteasome degradation of Stat3 contributes to the Ha-rasV12 overexpression-induced morphologic change. Although our current studies provided evidence that Ha-rasV12 overexpression downregulates Stat3 through proteosome degradation, it remains to be elucidated how Ha-rasV12 overexpression mediates targeting of Stat3 for degradation by proteasome.

To our knowledge, this is the first report to demonstrate that the levels of Stat3 expression are downregulated by Ha-ras overexpression, and we found this effect through proteosome degradation. In this study, we also found that Ha-rasV12 overexpression-downregulated Stat3 expression resulted from the activation of MEK/ERK pathway.Moreover, our data clearly indicate that the expression of Stat3 plays a critical role in Ha-rasV12-induced morphologic change in NIH3T3 cells.

Abbreviations

ERK

extracellular-regulated kinase

IPTG

isopropyl β-D-thiogalactopyranoside

JNK

Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

NRK

normal rat kidney

PI3K

phosphatidylinositol 3-kinase

Stat3

signal transducer and activator of transcription 3

Footnotes

1

This work was supported by grants from the Ministry of Education Program to Promote Academic Excellence in Universities (91-B-FA09-1-4) and the National Science Council (96-2628-B-006-003-MY3).

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