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J. Biol. Chem., Vol. 275, Issue 49, 38371-38377, December 8, 2000

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Cell Adhesion and Focal Adhesion Kinase Regulate Insulin Receptor Substrate-1 Expression^*

Patricia Lebrun^§, Véronique Baron^¶, Christof R. Hauck^**, David D. Schlaepfer, and Emmanuel Van Obberghen

From  INSERM U145, Institut Fdratif de Recherche 50, Avenue de Valombrose, 06107 Nice Cédex 2, France and the  Department of Immunology, Scripps Research Institute, La Jolla, California 92037

Received for publication, July 12, 2000, and in revised form, August 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrins are transmembrane receptors involved in interactions between cells and extracellular matrix proteins. Here we show that cell adhesion regulates insulin receptor substrate-1 (IRS-1) mRNA synthesis. When fibroblasts are held in suspension, lower levels of IRS-1 mRNA, but not of IRS-2 mRNA, are detected, and this effect is due to the negative regulation of IRS-1 transcription rather than to decreased mRNA stability. Upon fibronectin- or vitronectin-mediated integrin stimulation, the level of IRS-1 mRNA was restored within 4 h. The focal adhesion kinase (FAK) is known to be activated upon integrin stimulation, and we found that IRS-1 was not expressed in FAK^/ cells. Stable re-expression of epitope-tagged FAK in FAK^/ fibroblasts (DA2 cells) restored normal levels of IRS-1 expression, confirming that IRS-1 mRNA expression is regulated by FAK. It is known that integrins activate the JNK pathway. However, in adherent FAK^/ cells, we failed to detect activation of JNK, whereas JNK was stimulated in DA2 cells. This confirms the role of FAK in integrin-induced JNK stimulation. FAK-independent stimulation of JNK with anisomycin treatment both in FAK^/ cells and in suspended FAK^+/+ cells confirmed that IRS-1 mRNA transcription can be partially regulated by JNK. We suggest that integrins can modulate insulin and insulin-like growth factor-1 signaling pathways by regulating the levels of IRS-1 in cells and that FAK-mediated signaling to JNK is one pathway involved in this process.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrins are involved in several biological functions such as angiogenesis, wound repair, prevention of apoptosis, and cell migration and adhesion (1-6). Various intracellular events are induced by adhesion. For example, integrins regulate the intracellular concentrations of ions such as Ca²⁺, H⁺, and K⁺; activate several kinases like lipid kinases (phosphatidylinositol (PI)¹ 3-kinase and PI 5-kinase) and protein kinases; control the activity of phospholipases; and induce cytoskeleton rearrangements (7-9). It has been demonstrated that integrins can also influence the expression of several proteins. For instance, _IIb3 integrins have a positive effect on Bcl3 protein translation in platelets (10). Moreover, integrins activate the synthesis of platelet-derived growth factor B subunit mRNA in human monocytes (11). This correlates with high levels of c-Jun, c-Fos, and EGR2 mRNAs. In addition, adhesion of porcine vascular smooth muscle cells to fibronectin leads to the synthesis of insulin-like growth factor-binding protein-5 (12). This effect is inhibited by Arg-Gly-Asp (RGD) peptide and echistatin, which antagonize integrins. Recently, it has been shown that integrins regulate the interleukin-8 mRNA level in polymorphonuclear neutrophils (13). ₂ integrin aggregation was found to induce de novo synthesis and secretion of biologically active interleukin-8.

There are also several examples showing that integrins modulate the activity of transcription factors. In primary mouse mammary epithelial cells, the extracellular matrix is required for the prolactin-induced activation of STAT5 (signal transducer and activator of transcription 5) (14). In endothelial cells, the engagement of _v3 integrins by the extracellular matrix protein osteopontin rapidly increases nuclear factor-B activity and induces the translocation of the nuclear factor-B p65 form to the nucleus (15). In osteoclasts, activation of ₂ integrins is involved in the transcription of the gene encoding osteocalcin in response to ascorbic acid (16). Several studies have demonstrated that integrins activate JNK (17-20). Moreover, it has been shown recently that adhesion of NIH3T3 cells to fibronectin induces activation not only of Jun kinase, but also of the transcription factor AP-1 (21). Using a dominant-negative form of FAK, the study demonstrated that the effect depends on the activation of FAK and of the proto-oncogene c-src (21). In addition, FAK signaling is critical in the shear stress-induced stimulation of JNK and ERK in bovine aortic endothelial cells (22). FAK is one of the proteins implicated in the integrin signaling pathway. Following integrin engagement, FAK is activated and phosphorylated on tyrosine 397, which serves as a docking site for c-src (23). This association leads to further phosphorylation of FAK and allows its interaction with several signaling molecules such as the p85 subunit of PI 3-kinase, Grb2 (growth factor receptor-bound protein-2), and p130^CAS (Crk-associated substrate) (24-26). p130^CAS and Crk could also be involved in activation of the JNK pathway induced by several stimuli (21, 27, 28).

Since it has been demonstrated that integrins potentiate the insulin and IGF-1 signaling pathways, we studied the role of adhesion in the expression of IRS-1, a protein implicated in the intracellular cascade induced by insulin and IGF-1. We show here that in mouse fibroblasts, integrins regulate the expression of IRS-1, a major substrate of insulin/IGF-1 receptors and a central element of their signaling pathway. Furthermore, we provide evidence that FAK and JNK are key transducers of this effect of integrins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Culture media were from Life Technologies, Inc. [-³²P]dCTP and [-³²P]dATP were from ICN Pharmaceuticals (Costa Mesa, CA). Triton X-100, Nonidet P-40, leupeptin, benzamidine, pepstatin, and anisomycin were from Sigma. Aprotinin was from Bayer Pharma (Puteaux, France), and phenylmethylsulfonyl fluoride was from Serva (Heidelberg, Germany). Protein A was from Amersham Pharmacia Biotech (Uppsala, Sweden). Antiserum to IRS-1 was prepared in our laboratory and was raised against a synthetic peptide corresponding to the C-terminal sequence comprising amino acids 1223-1235 of rat IRS-1. Antibody to IRS-2 was kindly provided by M. F. White (Joslin Diabetes Center, Boston, MA). Antibody to FAK was prepared in our laboratory by immunizing rabbits with a synthetic peptide comprising amino acids 392-406. Antibody to IR (C19) was from Santa Cruz Biotechnology (Santa Cruz, CA), and antibody to phosphorylated JNK/SAPK was from New England Biolabs Inc. (Beverly, MA). Oligonucleotides were from Life Technologies, Inc. (Paisley, Scotland).

Cell Culture-- Wild-type (FAK^+/+) and knockout (FAK^/) fibroblasts were isolated from wild-type and knockout embryos generated by Ilic et al. (29). The stably transfected cells (DA2) have been described elsewhere (30, 31). Cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum at 37 °C and 5% CO₂.

Cell Adhesion Assay-- Cell culture dishes were coated with fibronectin (10 µg/ml) or vitronectin (2 µg/ml) in phosphate-buffered saline at 4 °C overnight. Dishes were rinsed twice with phosphate-buffered saline and warmed for 1 h at 37 °C before use.

Fibroblasts were maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum. Confluent cells were detached with trypsin, which was eliminated by centrifugation. Cells were resuspended in 10% fetal calf serum medium and kept in suspension for the indicated times. They were plated on coated dishes for increasing periods. Non-adherent cells were removed, and total RNA was extracted.

Northern Blotting-- Total RNA was extracted from cells using Trizol reagent (Life Technologies, Inc.). 15 µg of total RNA were denatured with formaldehyde/formamide and resolved by electrophoresis on 1.2% agarose gel containing formaldehyde. RNA was then transferred onto a Hybond membrane (ICN Pharmaceuticals), which was hybridized at 42 °C for 18 h. The probes used were cDNA fragments of IRS-1 (1-2.8 kb), IRS-2 (2.9-3.9 kb), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 0.6 kb). They were labeled with [-³²P]dCTP using a random primer labeling kit (Rediprime kit) and purified with a ProbeQuant kit (both from Amersham Pharmacia Biotech). Hybridization of 18 S RNA was performed using the oligonucleotide 5'-ACG GTA TCT GAT CGT CTT CGA ACC-3' labeled with [-³²P]dATP using T4 kinase (Life Technologies, Inc.)

Reverse Transcription-PCR Analysis-- Cells were incubated with actinomycin D (5 µg/ml) for 1-6 h and then lysed, and total RNA was extracted. Reverse transcription was performed on 5 µg of total RNA using the mouse mammary leukemia virus enzyme and oligo(dT) primers. The reverse transcription products were then used as templates for PCR amplification using gene-specific primers: for IRS-1, 5'-ACCATGGGGACAAGCCCGGCG-3' and 5'-GGGGCTGCTGGTGTTGGAATC-3'; and for -actin, 5'-GTGGGGGCGCCCCAGGCACCA-3' and 5'-TCCTTAATGTCACGCACGATTTC. The linear range of the PCR was determined by checking the amount of PCR products after various cycles of amplification to allow comparison between different samples. PCR products were separated on a 1% agarose gel and analyzed by ethidium bromide incorporation.

Immunoprecipitation and Western Blotting-- Cells were washed with buffer A (50 mM Hepes (pH 7.5), 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na₄P₂O₇, and 2 mM NaVO₄) and then lysed for 15 min on ice with buffer A supplemented with 1% (v/v) Nonidet P-40, 20 mM leupeptin, 4 mM benzamidine, 2 mM pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 100 units/ml aprotinin. After centrifugation at 4 °C for 15 min, cell lysates (800 µg of proteins) were added to antibodies (anti-IRS-1, serum dilution of 1:50; anti-IRS-2, serum dilution of 1:200; and anti-IR, 1 µg of purified Ig) preadsorbed on protein A-Sepharose. Immunoprecipitation was performed at 4 °C for 3 h. Pellets were then washed three times with buffer A containing 0.1% Nonidet P-40 and resuspended in Laemmli buffer (32). Immunoprecipitated proteins or whole lysates were analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions, and proteins were transferred to an Immobilon membrane (polyvinylidene difluoride; Millipore Corp.) in 25 mM Tris, 192 mM glycine, and 20% methanol (pH 8.3). The membrane was blocked with TBS (10 mM Tris (pH 7.4) and 140 mM NaCl) containing 5% (w/v) bovine serum albumin for 1 h and incubated with antibodies to IRS-1 (1:1000), IRS-2 (1:1000), IR (0.7 µg/ml), FAK (1:200), HA tag (1:4000), or phosphotyrosine (1:2000) for 60 min at room temperature and with antibodies to phospho-JNK/phospho-SAPK (1:1000) or JNK/SAPK for 1 night at 4 °C. The membrane was washed four times with TBS containing 1% (v/v) Nonidet P-40 and then twice with TBS. Finally, the membrane was incubated with horseradish peroxidase-conjugated antibodies (Dako AS, Glostrap, Denmark) for 1 h at room temperature, washed four times with TBS containing 1% (v/v) Nonidet P-40 and twice with TBS, revealed with chemiluminescent substrate (ECL, Pierce), and exposed to sensitive films.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IRS-1 mRNA Is Decreased in Suspended Fibroblasts-- In this study, we investigated the role of cellular adhesion in the regulation of IRS-1 expression. To this end, FAK^+/+ fibroblasts either were kept attached or were detached from plates and kept in suspension. The two series of cells were then compared. Total RNA was extracted after different times of suspension, and IRS-1 mRNA was analyzed by Northern blotting. The results are presented in Fig. 1. After 2 h of suspension, the level of IRS-1 mRNA (normalized to amounts of 18 S rRNA) decreased (~20% left) and remained very low at 4 h (~12% left). IRS-2 mRNA, which was studied in parallel, was not down-regulated in suspended fibroblasts, but on the contrary, seemed to be up-regulated. The 18 S rRNA quantification was used as a loading control. Amounts of GAPDH mRNA (normalized to amounts of 18 S rRNA) were similar in suspended and adherent fibroblasts. Thus, disruption of cellular adhesion results in a marked loss of IRS-1 mRNA in fibroblasts.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   IRS-1 mRNA expression in adherent and suspended fibroblasts. FAK+/+ fibroblasts were left adherent or were detached from plates and kept in suspension for 2 or 4 h. Total RNA was extracted, resolved on a formaldehyde gel, and subjected to Northern blotting. IRS-1, IRS-2, and GAPDH mRNAs were revealed using IRS-1 (1-2.8 kb), IRS-2 (2.9-3.9 kb), and GAPDH (0.6 kb) cDNAs, respectively. The 18 S rRNA was revealed with an oligonucleotide. Upper panel, autoradiogram of the Northern blot; lower panel, autoradiogram of three experiments scanned and quantified using NIH Imager. The amounts of IRS-1 were normalized to amounts of 18 S rRNA and are expressed as a percentage of the adherent conditions.

Stability of IRS-1 mRNA Is Similar in Suspended and Adherent Fibroblasts-- Since the level of IRS-1 mRNA was decreased in suspended fibroblasts, we next examined if this was due to decreased mRNA stability. To do this, suspended and adherent FAK^+/+ fibroblasts were treated with actinomycin D (5 µg/ml) for 45 min, 1.5 h, and 3 h to block transcription. Total RNA was extracted and analyzed by Northern blotting. As shown in Fig. 2, the levels of IRS-1 and IRS-2 mRNAs were already decreased after 45 min of actinomycin D treatment. After 3 h, both were undetectable. We calculated that the half-life of IRS-1 mRNA was 1 and 1.5 h in adherent and suspended fibroblasts, respectively. A similar degradation rate was observed for IRS-2 mRNA. The GAPDH and 18 S RNA amounts remained unchanged even after 3 h of actinomycin D treatment in suspended and adherent fibroblasts. We conclude that the decreased level of IRS-1 mRNA observed in suspended cells is not due to decreased mRNA stability.

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Stability of IRS-1 mRNA in suspended and adherent fibroblasts. FAK+/+ fibroblasts detached from plates or left adherent were incubated with actinomycin D (5 µg/ml) for 45 min to 3 h. Total RNA was extracted and analyzed by Northern blotting. IRS-1, IRS-2, and GAPDH mRNAs were revealed using IRS-1 (1-2.8 kb), IRS-2 (2.9-3.9 kb), and GAPDH (0.6 kb) [-32P]dCTP-labeled cDNAs, respectively. The 18 S rRNA was revealed with a [-32P]dATP-labeled oligonucleotide. An autoradiogram of a representative experiment is shown.

Cell Adhesion to Fibronectin and Vitronectin Induces Synthesis of IRS-1 mRNA-- To further investigate the role of integrins in the regulation of IRS-1 expression, we measured the synthesis of IRS-1 mRNA upon activation of integrins. To this end, FAK^+/+ fibroblasts were detached from plates and kept in suspension for 2 h. They were then replated on fibronectin or vitronectin, both of which engage the ₅₁ or _v3 integrins, respectively. Total RNA was extracted after 1, 2, or 4 h of adhesion, and IRS-1 mRNA was revealed by Northern blotting (Fig. 3). Again, IRS-1 mRNA was decreased after 2 h of suspension. Importantly, the level of IRS-1 mRNA was restored after 4 h of adhesion to fibronectin as well as to vitronectin. The GAPDH and 18 S RNA amounts were unchanged. These experiments show that activation of integrins by fibronectin or vitronectin induces the synthesis of IRS-1 mRNA. Since the tyrosine kinase FAK is one of the major proteins activated in adherent cells, but inactivated in suspended cells, we next studied the potential role of FAK in the regulation of IRS-1 expression.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Expression of IRS-1 mRNA in fibroblasts replated on fibronectin and vitronectin. FAK+/+ fibroblasts were left attached or were detached and kept in suspension for 2 h. Cells were replated for 1, 2, or 4 h on fibronectin (10 µg/ml) or vitronectin (2 µg/ml). Total RNA was extracted and subjected to Northern blotting. The mRNAs of interest were revealed as described in the legend to Fig. 1. A representative autoradiogram is shown.

Expression of IRS-1 mRNA Is Abolished in FAK^/ Fibroblasts-- To investigate the role of FAK in the control of IRS-1 mRNA levels, we used FAK knockout cells (FAK^/) (29). Total RNA was extracted from FAK^/, FAK^+/+, and NIH3T3 fibroblasts. mRNAs were analyzed by Northern blotting. As expected, the Northern blot revealed IRS-1 mRNA in NIH3T3 fibroblasts and FAK^+/+ cells (Fig. 4, upper panel). However, no IRS-1 mRNA was detected in FAK^/ cells. On the contrary, the levels of IRS-2 mRNA were the same in FAK^/ and FAK^+/+ cells. Note that GAPDH mRNA levels were also the same in both cell lines and that IRS-2 mRNA was also expressed in NIH3T3 fibroblasts. In summary, knockout of the gene encoding FAK leads to reduced synthesis of IRS-1 mRNA, but has no effect on IRS-2 mRNA.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   IRS-1 mRNA expression in FAK/ fibroblasts. Upper panel, total RNA was extracted from confluent knockout fibroblasts (FAK/) and from wild-type fibroblasts (FAK+/+ and NIH3T3). IRS-1, IRS-2, and GAPDH mRNAs were revealed by Northern blotting using specific cDNAs corresponding to IRS-1, IRS-2, and GAPDH. A representative autoradiogram out of two experiments is shown. Lower panel, knockout and wild-type fibroblasts were treated with actinomycin D (5 µg/ml) for 1, 2, 4, or 6 h. After extraction of total RNA, reverse transcription was performed on 5 µg. cDNAs obtained were used to perform a co-amplification of IRS-1 and -actin with specific oligonucleotides. PCR products were resolved on a 1% agarose gel and analyzed for ethidium bromide incorporation.

We next determined if there was a decreased stability of the IRS-1 mRNA in FAK^/ cells. As there were very low amounts of IRS-1 mRNA in these cells, a method more sensitive than Northern blotting was used, i.e. reverse transcription-PCR. Cells were treated with actinomycin D (5 µg/ml) for the indicated periods of time to block transcription. Total RNA was extracted and subjected to reverse transcription-PCR as described under "Experimental Procedures." As shown in Fig. 4 (lower panel), the level of IRS-1 mRNA rapidly decreased in FAK^+/+ cells, consistent with the half-life calculated from results shown in Fig. 2. The amount of IRS-1 mRNA was strongly reduced in FAK^/ cells compared with FAK^+/+ cells. Due to the very low level of IRS-1 mRNA in FAK^/ cells, it was not possible to calculate its half-life. -Actin mRNA was stable even after 6 h of actinomycin D treatment and was the same in both cell lines. As a whole, our results indicate that FAK may be required for synthesis of IRS-1 mRNA.

Expression Level of the IRS-1 Protein in FAK^/ Cells-- Protein expression of IRS-1, IRS-2, and IR was determined in FAK^/ and FAK^+/+ fibroblasts. To do this, FAK^/ or FAK^+/+ cells were lysed, and immunoprecipitation of IRS-1, IRS-2, and IR was performed. Immunopurified proteins were analyzed by Western blotting using antibodies to IRS-1, IRS-2, and IR (Fig. 5). As expected, the IRS-1 protein was undetectable in FAK^/ cells, whereas no difference in the expression of IRS-2 and IR was observed between the two cell lines. In this experiment, FAK was correctly expressed in the wild-type cells, whereas no FAK was present in the mutant cells (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Expression of IRS-1 protein in FAK/ fibroblasts. Immunoprecipitation (IP) of IRS-1, IRS-2, and IR was carried out on lysates from FAK/ and FAK+/+ cells. Immunoprecipitates were analyzed by Western blotting using antibodies to IRS-1, IRS-2, and IR. Results were revealed with a chemiluminescence detection system (Pierce). The autoradiogram shown is representative of three comparable experiments.

To further establish the role of FAK in IRS-1 gene expression, we investigated whether the expression of FAK in FAK^/ cells would restore IRS-1 protein expression. To do this, we used DA2 cells, which are FAK^/ cells stably transfected with an empty vector or a vector containing the cDNA encoding HA-tagged FAK (30). These cells were lysed, and immunoprecipitation of IRS-1 was performed using a specific antibody to IRS-1. Immunopurified proteins and total lysates were analyzed by Western blotting using antibodies to IRS-1 or FAK. FAK expression was checked in all cell lines using an antibody to FAK (Fig. 6B, middle panel). Due to the HA tag, FAK had an increased molecular mass in DA2 cells. An antibody to HA revealed that the protein observed in DA2 cells using the antibody to FAK corresponds to HA-tagged FAK (Fig. 6B, lower panel). We found that IRS-1 expression was restored in the stably transfected DA2 cell line that expresses FAK (Fig. 6, A and B, upper panel). So far, we cannot explain why IRS-1 in DA2 cells seems to have an increased electrophoretic mobility compared with IRS-1 in FAK^+/+ cells. Taking the results of Figs. 5 and 6 together, we suggest that FAK plays a positive role in the regulation of IRS-1 protein expression, but does not appear to be involved in IRS-2 or IR expression.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Restored expression of IRS-1 protein in FAK/ cells stably transfected with FAK. Two representative experiments are presented. A, total lysates were extracted from knockout fibroblasts (FAK/), knockout fibroblasts stably transfected with an empty vector (FAK/;vect), knockout fibroblasts stably transfected with FAK (DA2), and wild-type fibroblasts (FAK+/+). Immunoprecipitation (IP) of IRS-1 was performed on lysates, and immunocomplexes were separated on a 7.5% polyacrylamide gel and analyzed by Western blotting. B, immunoprecipitation of IRS-1 was performed on one part of the lysates. Immunoprecipitates and a fraction of whole lysates were separated on a minigel and analyzed by Western blotting using antibodies to IRS-1 and to FAK and the HA tag, respectively.

Integrins Do Not Activate JNK in FAK^/ Cells-- It has previously been demonstrated in several cell types that integrins can activate JNK and that FAK is implicated in this process (20, 21). Therefore, we examined whether this occurred also in FAK^/ cells. To this end, FAK^/ cells were serum-starved and kept in suspension for 1 h. Cells were then replated on fibronectin for 10 min, 30 min, 1 h, and 4 h. Cells were lysed, and proteins were separated on a large-size polyacrylamide gel. Western blotting was performed using antibodies to activated JNK. As shown in Fig. 7 (upper panels), JNK was not activated in FAK^/ cells in response to integrin engagement. Note that suspension of cells led to a weak stimulation of JNK. This effect could be caused by the engagement of stress signaling pathways due to serum depletion and suspension and is likely to be unrelated to the integrin signaling pathway. To document the role of FAK in integrin-induced JNK activation, we performed the same experiment in DA2 cells. The results presented in Fig. 7 (lower panels) demonstrate that the re-expression of FAK in FAK^/ cells is sufficient to restore the activation of JNK by integrins, which reaches a peak within 10 min and then fades out. Western blotting carried out with antibodies to JNK revealed similar levels of proteins under all conditions.

View larger version (48K): [in this window] [in a new window]   Fig. 7.   FAK contributes to fibronectin-stimulated JNK activation. FAK/ or FAK-re-expressing DA2 cells were either serum-starved (0.5% serum for 24 h), detached by limited trypsin/EDTA treatment, and suspended for 1 h or replated on fibronectin-coated (10 µg/ml) dishes for the times indicated in the absence of serum. Equal amounts (100 µg) of whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by anti-phospho-activated JNK blotting (Promega). Blots were reprobed with polyclonal antibodies to JNK-1 (Santa Cruz Biotechnology) to verify equal amounts of JNK-1 protein.

Activation of the JNK Pathway Is Implicated in IRS-1 mRNA Expression-- To study the involvement of JNK in the synthesis of IRS-1 mRNA, FAK^/ cells were treated with anisomycin (50 ng/ml) for the indicated times to stimulate JNK. Total RNA was extracted, and IRS-1 mRNA was revealed by Northern blotting. Activation of JNK by anisomycin was tested after 30 and 60 min of stimulation. Proteins were analyzed by Western blotting, and activated JNK and SAPK were revealed using a specific antibody. As illustrated in Fig. 8 (lower panels), JNK and SAPK were fully activated by anisomycin in FAK^/ cells after 30 min of treatment, and the stimulation started to decrease after 60 min. Fig. 8 (upper panel) shows that IRS-1 mRNA levels increased in a time-dependent manner in FAK^/ cells. However, the amount of IRS-1 mRNA in FAK^/ cells after 5 h of stimulation with anisomycin was still 2-fold smaller than that in FAK^+/+ cells. Therefore, it appears that activated JNK only partially induces IRS-1 mRNA synthesis in the absence of FAK. Taken together, these results demonstrate that JNK is one of the pathways implicated in IRS-1 mRNA synthesis.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Involvement of the JNK pathway in IRS-1 mRNA expression in adherent FAK/ cells. Adherent FAK/ cells were incubated with anisomycin (50 ng/ml) for 2 or 5 h. Upper panel, total RNA was extracted, and IRS-1, GAPDH, and 18 S RNAs were analyzed by Northern blotting. Lower panels, cells were lysed after 30 and 60 min of treatment with anisomycin. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and activated JNK/SAPK was revealed using specific antibodies. Blots were reprobed with monoclonal antibodies to JNK/SAPK. Representative autoradiograms are shown.

To further establish the role of JNK in integrin-induced IRS-1 mRNA synthesis, we determined if JNK activation could restore the IRS-1 mRNA level in suspended cells. To this end, FAK^+/+ cells were treated with anisomycin at the indicated concentrations for 30 min. Cells were then kept in suspension for 2 h in the presence of anisomycin, and total RNA was extracted. IRS-1 mRNA expression was revealed by Northern blotting. The results are presented in Fig. 9 (upper panel). As previously demonstrated, the IRS-1 mRNA level decreased after 2 h of suspension. However, a dose-dependent increase in IRS-1 mRNA was observed in suspended cells treated with anisomycin. At 50 ng/ml anisomycin, the increase in IRS-1 mRNA was maximal, and the level of IRS-1 mRNA was very similar to the one detected in adherent cells. Activation of JNK by anisomycin was tested on whole cell lysates after 2 h of suspension under all conditions. Proteins were revealed by Western blotting using antibodies to phospho-JNK/phospho-SAPK and JNK/SAPK. As shown in Fig. 9 (lower panels), treatment with anisomycin induced a dose-dependent activation of JNK and SAPK. Interestingly, it seems that in suspended cells, anisomycin treatment slightly induced the expression of JNK. Therefore, it is possible that this effect contributes to the positive regulation of IRS-1 mRNA synthesis observed upon anisomycin treatment.

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Involvement of the JNK pathway in IRS-1 mRNA expression in suspended FAK+/+ cells. FAK+/+ cells were pretreated with increased concentrations of anisomycin for 30 min and then put in suspension in the presence of the indicated amounts of anisomycin for 2 h. Upper panel, total RNA was extracted, and IRS-1, GAPDH, and 18 S RNAs were analyzed by Northern blotting. Lower panels, cells were lysed after 2 h of suspension and anisomycin treatment. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and activated JNK/SAPK was revealed using specific antibodies. Blots were reprobed with monoclonal antibodies to JNK/SAPK. Representative autoradiograms are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies have demonstrated the existence of a cross-talk between signaling pathways used by integrins and those used by growth factors (8). It is generally believed that adhesion is required for growth factors to induce the full array of their intracellular effects. More specifically, there have been examples for integrin regulation of insulin and IGF-1 signaling pathways (33-35). Insulin and IGF-1 receptors are tyrosine kinase receptors, which are activated and autophosphorylated upon ligand binding. This leads to the tyrosine phosphorylation of the docking proteins IRS-1 and IRS-2 and to their subsequent interaction with several SH2 domain-containing proteins such as the regulatory subunit of PI 3-kinase, Grb2, SHP-2 (SH2 domain-containing phosphotyrosine phosphatase-2), and Nck (36, 37). Insulin and IGF-1 affect several cellular events, including metabolism, cell growth and differentiation, and apoptosis (38, 39).

The cellular effects of IGF-1 such as DNA and protein synthesis are potentiated by engagement of _v3 integrins in smooth muscle cells (40), whereas in Chinese hamster ovary cells, ₅₁ integrins synergize with the insulin signal (33, 34). These effects may be mediated by increased tyrosine phosphorylation of the receptors and their substrates. In addition, human pancreatic carcinoma cells overexpressing _v3 integrins show a 2.5-fold increased DNA synthesis in response to insulin when cells are plated on vitronectin (41). It has been demonstrated in Rat1 cells and in human pancreatic carcinoma cells that, upon insulin stimulation, both IRS-1 and IR interact with _v3 integrins (42).

In the context of cross-talk between signaling pathways used by integrins and those used by insulin and IGF-1, our work provides evidence for the requirement of integrins for IRS-1 expression. Indeed, disruption of adhesion induces a fast down-regulation of IRS-1 mRNA, which drops to very low levels within 2 h. Conversely, integrin engagement on fibronectin or vitronectin restores the level of IRS-1 mRNA within 4 h. The molecular mechanisms implicated in this regulation remain to be defined. It has been demonstrated that engagement of integrins induces the recruitment and activation of tyrosine kinases such as FAK and Src (43). The tyrosine kinase FAK, which is a key player in integrin signaling, is inactivated in suspended fibroblasts, but becomes activated upon cell adhesion. Therefore, this protein is a likely candidate for a regulator of IRS-1 gene expression. We used FAK^/ cells to investigate FAK putative involvement in the control of IRS-1 mRNA levels. Interestingly, IRS-1 mRNA and protein were not expressed in FAK knockout cells, suggesting a role for FAK. Complete FAK deficiency in FAK^/ animals is lethal at an early stage of development due to a pronounced defect of mesoderm maturation (29). No profound protein down-regulation has been observed in these cells so far. In fact, protein expression of the FAK-related tyrosine kinase PYK2 is increased in FAK^/ cells and compensates for FAK absence regarding the activation of ERK2, but not cell migration (44). However, PYK2 does not appear to be able to maintain IRS-1 expression in FAK^/ cells. Moreover, ectopic expression of FAK in FAK^/ cells is sufficient to restore the synthesis of IRS-1 to a normal level. Since FAK knockout cells show a certain degree of disorganization of the cortical cytoskeleton, we cannot exclude that this could participate in the pathways leading to regulation of IRS-1 expression in FAK^/ cells.

Previous studies have demonstrated that integrin engagement activates JNK and that FAK can be implicated in this process (20, 21). Accordingly, our adhesion experiments showed that in FAK re-expressing DA2 cells, integrins strongly and transiently activated JNK, whereas JNK stimulation was not seen in FAK^/ cells replated on fibronectin (Fig. 7). However, a weak amount of phosphorylated JNK was detected in suspended FAK^/ and FAK^+/+ cells. This activation could be due to stress induced by suspension and serum depletion and is unrelated to integrin-mediated effects (21). Treatment of adherent FAK^/ cells and suspended FAK^+/+ cells with the activator of the JNK pathway, anisomycin, partially restores IRS-1 mRNA expression. In fact, it seems that anisomycin not only activates JNK, but induces JNK expression in suspended fibroblasts. Taken all together, our results show that IRS-1 mRNA expression strongly correlates with increased JNK signaling. Since anisomycin treatment only partially restores the IRS-1 mRNA level, additional pathways are likely to be implicated in the regulation of IRS-1 expression. It is well known that integrins activate the MAPK pathway and that FAK is involved in this process. To investigate the potential role of MAPK in IRS-1 mRNA synthesis, we overexpressed a constitutively active form of MEK (the upstream activator of MAPK) in FAK^/ and FAK^+/+ cells. In these cells, no synthesis of IRS-1 mRNA was detected (data not shown). This suggests that the MAPK pathway is not implicated in the regulation of IRS-1 expression. Moreover, it has been demonstrated that PI 3-kinase is activated in response to integrins and that the interaction between FAK and p85 is involved in this process. However, our preliminary experiments using the specific PI 3-kinase inhibitor wortmannin suggest that the PI 3-kinase pathway is not implicated in the regulation of IRS-1 synthesis by integrins (data not shown).

Very few things are known about transcriptional regulation of the IRS-1 gene. Its promoter lacks typical CAAT and TATA boxes, but contains nine potential Sp1-binding sites, consistent with the structure of a housekeeping gene (45). It also contains AP-1-, AP-2-, and CAAT/enhancer-binding protein-binding sites; an E-box; and several hormone response elements such as the glucocorticoid response element and the progesterone response element (46). It has been reported that integrins regulate the activities of JNK and of the AP-1 transcription factor and that FAK is implicated in this control (21). Together with our findings presented here, these data suggest that integrins regulate the transcription of the IRS-1 gene via FAK-mediated JNK activation.

A remarkable finding of our work is that integrins quite differently affect the expression level of IRS-1 mRNA versus IRS-2 mRNA. Indeed, in adherent fibroblasts, integrins positively regulate IRS-1 gene expression, but seem to lead to a decrease in IRS-2 mRNA expression. Moreover, IRS-1 expression is abolished in FAK knockout cells, whereas the level of IRS-2 is unchanged. IRS-1 and IRS-2 are thought to play distinct roles in the biological effects mediated by insulin and IGF-1. Indeed, several studies suggest that IRS-1 preferentially mediates the mitogenic effects of insulin and IGF-1, whereas IRS-2 appears to be more involved in generating the metabolic responses of insulin (47-50). Considering this, we suggest that the positive control of IRS-1 expression (but not IRS-2 expression) by integrins could alter specific responses. However, a very recent report provides evidence that IRS-1 and IRS-2 can share biological effects, but that they exert tissue-specific roles (51). IRS-1 would play a prominent role in skeletal muscle, whereas IRS-2 would be the dominant player in liver. In such a model, integrins would have a prominent role in the regulation of the insulin response in skeletal muscle and would have a less pronounced impact in insulin signaling in the liver.

Another interesting possibility to consider is that IRS-1 participates in signaling mediated by FAK in response to integrin engagement independently of the insulin or IGF-1 circuitry. Indeed, we have demonstrated that FAK interacts directly with IRS-1 (35). This association leads to tyrosine phosphorylation of IRS-1 and its interaction with SH2 domain-containing proteins such as p85, Grb2, and SHP-2. Finally, FAK activates IRS-1-associated PI 3-kinase. In this context, down-regulation of IRS-1 expression in suspended cells may act as a negative feedback loop in the integrin signaling pathway.

To conclude, a picture is emerging that shows that cell adhesion is required to allow optimal functioning of integrated cell systems. Our present study and previous reports (34, 40, 41) suggest that cell adhesion is required for the full use of IRS-1 signaling machinery by insulin and IGF-1.

	ACKNOWLEDGEMENT

We thank D. Ilic for the generous gift of FAK^/ and FAK^+/+ cells.

	FOOTNOTES

^* This work was supported in part by INSERM, Université de Nice-Sophia-Antipolis, and by the Ligue Nationale contre le Cancer (Axe Oncogenèse).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a student fellowship from the Ligue Nationale contre le Cancer.

^¶ To whom correspondence should be addressed. Tel.: 33-4-93-37-77-96; Fax: 33-4-93-81-54-32; E-mail: vbaron@skcc.org.

^** Supported by Postdoctoral Fellowship HA 2856/1-1 from the Deutsche Forschungsgemeinschaft.

Supported by United States Public Health Service Grant R29 CA75240 from NCI, National Institutes of Health.

Published, JBC Papers in Press, August 30, 2000, DOI 10.1074/jbc.M006162200

	ABBREVIATIONS

The abbreviations used are: PI, phosphatidylinositol; JNK, c-Jun NH₂-terminal kinase; FAK, focal adhesion kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; IGF-1, insulin-like growth factor-1; IR, insulin receptor; IRS, insulin receptor substrate; SAPK, stress-activated protein kinase; kb, kilobase pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; TBS, Tris-buffered saline; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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