Integrin signaling and cell spreading alterations by

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Mar 21, 2009 - general PKC inhibitors such as Gö6976 and Gö6983 and Bisindo- lylmaleimide, did not affect the migration of chick wing bud mesenchymal ...
Biochimie 91 (2009) 624–631

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Research paper

Integrin signaling and cell spreading alterations by rottlerin treatment of chick limb bud mesenchymal cells Young-Ae Choi a, Dong-Kyun Kim b, Shin-Sung Kang a, Jong-Kyung Sonn a, Eun-Jung Jin b, c, * a

Department of Biological Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea Department of Biological Sciences, College of Natural Sciences, Wonkwang University, Iksan, Chunbuk 570-749, Republic of Korea c Institute of Biotechnology, Wonkwang University, Iksan, Chunbuk 570-749, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 January 2009 Accepted 12 March 2009 Available online 21 March 2009

Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. Prechondrogenic condensation, in which mesenchymal cells aggregate, requires cell migration and proliferation. However, the molecular mechanisms promoting this aggregation remain to be elucidated. Here, we report that rottlerin suppresses migration and cell surface expression of integrin b1 in chondrogenic progenitors. Perturbation of integrin b1 function using an anti-integrin b1 blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by rottlerin treatment. Cell treatment with PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, suppressed cell migration in a wound-healing assay. Cells treated with rottlerin showed decreased phosphorylation of Akt, independent of PKCd inhibition. In addition, an Akt inhibitor suppressed the migration of chick limb bud mesenchymal cells. Taken together, our results point to the novel finding that rottlerin may act as a negative regulator for cell migration, an essential step for prechondrogenic condensation, by regulating integrin b1 signaling at focal adhesion complexes via modulation of Akt activity. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Rottlerin Integrin b1 Wound-healing assay Chondrogenic differentiation Cellular condensation

1. Introduction Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes [1]. The development of the endochondral skeleton begins with a process of prechondrogenic condensation [2–6] and intimate cell–cell interactions promote this condensation [7,8]. Condensation begins by chondroprogenitor cell–cell homophilic adhesions [3,9] with an increased number of intercellular contacts occurring through gap junctions [10]. Hyaluronic acid, fibronectin and type I collagen all increase, and overt differentiation begins at the center of this precartilage with a subsequent increase in the production of type II collagen. Mesenchymal condensation results from either an increase of mitotic activity or from active cell movement causing an increase in

* Corresponding author at: Faculty of Biological Sciences, College of Natural Sciences, Wonkwang University, Iksan, Chunbuk 570-749, Republic of Korea. Tel.: þ82 63 850 6197; fax: þ82 63 857 8837. E-mail address: [email protected] (E.-J. Jin). 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.03.006

mesenchymal cell packing density without an increase in cell proliferation [3,9,10]. Thus, disruption of cell migration toward an aggregation center could alter the degree of precartilage condensation. It is known that a small family of Rho GTPases plays an important role in cell migration by transducing signals that regulate changes in actin and microtubule cytoskeletal networks [11]. However, any mutual regulatory linkages between cell migration and condensation are largely unknown. Rottlerin is a pharmacological inhibitor of protein kinase Cs (PKCs) with selectivity for PKCd [12] and rottlerin also inhibits the activities of a number of unrelated kinases, including Akt/PKB and CaMKs [13]. Recent reports have shown that rottlerin may possibly induce apoptosis in lung cancer, breast cancer, chronic lymphocytic leukemia and multiple myeloma cells [14–17]. Particularly rottlerin is known to regulate tumor cell migration [18]. In this study, we also found that rottlerin affect migration of chick limb mesenchymal cells during chondrogenesis. Even though migration and adhesion of limb mesenchymal cells are essential prerequisites for the formation of a cartilaginous template during endochondral skeletal development, the regulatory and signaling mechanism is not well established. Therefore, verifying the signaling mechanism in rottlerin-regulated cell migration led us to understand the mechanism

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of cellular condensation during chondrogenesis. In this study, we applied a high density (micromass) culture model system [19] that allowed condensation (i.e. increased cell density due to cell aggregation) to clarify a role for rottlerin during prechondrogenic condensation. We found a negative regulatory role for rottlerin during migration of chondrogenic progenitors, an essential step for prechondrogenic condensation. Rottlerin-regulated integrin b1 signaling in focal adhesion complexes through a protein kinase Cd-independent pathway. 2. Materials and methods 2.1. Cell culture and treatment Mesenchymal cells were derived from the distal tips of wing buds from Hamburger–Hamilton [20] stage 22/23 embryos of fertilized white Leghorn chicken eggs. The cells were micromasscultured as described previously [21]. Cells at 2  107 cells/ml were plated onto 35 or 60 mm dishes in 3 or 19 drops of 15 ml each. The cells were incubated for 1 h at 37  C under 5% CO2 to allow attachment and were then maintained in Ham’s F-12 medium containing 10% fetal bovine serum, 100 IU/ml penicillin, and 50 mg/ ml streptomycin (Gibco Invitrogen, Grand Island, NY). The cells were maintained in culture medium for the indicated time periods in the presence or absence of the following reagents: 1000 nM rottlerin, 10 mM Go¨6976, 10 mM Go¨6983, 10 mM Bisindolylmaleimide, 10 mM PP2, 500 nM Akt inhibitor IV (Chemicon, Temecula, CA) unless the concentrations were otherwise indicated.

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2.2. Functional blocking of integrin b1 Cells were incubated for 1 h with 2 mg/ml anti-b1 integrin blocking antibody (Chemicon), purified hamster IgM or vehicle. 2.3. Analysis of cell condensation and differentiation Chondrogenic differentiation was measured by Alcian blue staining of sulfated cartilage glycosaminoglycans (GAG). To demonstrate the deposition of cartilage matrix proteoglycans, representative cultures were collected at day 5 of incubation and stained with 0.5% Alcian blue 8GX, pH 1.0 [22,23]. Alcian blue bound to sulfated GAG was extracted with 6 M guanidine–HCl, and quantified by measuring the absorbance of the extracts at 600 nm. Binding of peanut agglutinin (PNA) was used as a specific marker for precartilage condensation. Briefly, cultures were rinsed twice with 0.02 M PBS, pH 7.2, fixed in methanol:acetone (1:1) for 1 min, air-dried, and then incubated with 100 mg/ml biotinylated PNA (Sigma) for 1 h. Bound PNA was visualized using the VECTASTAIN ABC and DAB substrate solution kit (Vector laboratories Inc., Burlingame, CA). 2.4. Apoptosis assay Apoptosis was analyzed by a flow cytometer (FACS calibure, Becton–Dickinson, France). To detect extent of propidium iodide, cells were excited at 488 nm and emission was observed at 585 nm.

Fig. 1. Rottlerin suppresses chondrogenesis. Wing mesenchymal cells were cultured at a density of 2  107 cells/ml and treated with varying concentrations of rottlerin (upper panel). Chondrogenesis was quantified by measuring the absorbance of bound Alcian blue at 600 nm (lower panel).

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Cell migration assay was adapted from a wounding assay as previously described [24]. Cells were cultured in growth media for 10 h prior to performing a migration assay. For the migration assay, an area on a plate was cleared of cells using a pipette tip. Next, the cultures were incubated for 15 h in growth media supplemented with various chemicals to allow migration. Cultures were photographed at zero-time and at 15 h after clearing. Cell migration was determined by measuring the difference between the cleared area prior to migration from the cleared area remaining after migration (area restored) using image J software (version 1.36b, freeware downloaded from http://rsb.info.nih.gov/ ij/, an image processing and analysis program developed at the National Institutes of Health, USA). Three independent experiments were performed. 2.6. Western blot analysis Total protein was prepared using RIPA buffer and Western blot analyses were performed as previously described [25]. Thirty micrograms total protein were loaded and resolved on a 10% SDSPAGE gel. Proteins were transferred to a nitrocellulose membrane and probed with the following primary antibodies: (p)Erk, (p)Akt, (p)FAK576 and (p)FAK925 (Cell Signaling Technology Inc., Danvers, MA, USA) and (p)Src (Invitrogen), PKC (Santa Cruz), FN (Sigma), integrin b1 (BD Science, Franklin Lakes, NJ, USA), (p)FAK397 (Covance, Princeton, NJ, USA), and (p)FAK861 (Abcam Inc., Cambridge, MA, USA). Membranes were washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz). Signal was detected by enhanced chemiluminescence (Pierce Biotechnology Inc., Rockford, MN). Three independent experiments were performed for all Western blot analyses. 2.7. siRNA and antisense oligonucleotide transfection To generate constructs encoding small interfering RNA (siRNA) for FAK-2, oligonucleotides containing 60 nucleotides were synthesized as shown in Scheme 1, where ‘‘FAK-2: sense’’ and ‘‘FAK2: antisense’’ represent 19 nucleotide sequences linked by a hair pin structure. The 19 nucleotide sequences for FAK-2 specific antisense were 50 -GGAAATATGAACTGAGAA T-30 . The oligonucleotides were annealed and then ligated into the pSUPER vector (Oligoengine, Seattle, WA) digested with BglII and XhoI. Positive clones from ampicillin resistant colonies were detected by the presence of an EcoRI–HindIII restriction fragment. Electroporation of mesenchymal cells with the siRNA-encoding vector was conducted using a square wave generator (BTX-830; Gentronics, San Diego, CA) with 20 ms, 200 square pulses. 5’-GATCCCC(FAK: sense)TTCAAGAGA(FAK: antisense)TTTTTA-3’ 3’-GGG(FAK: sense)AAGTTCTCT(FAK: antisense)AAAAATTCGA-5’ Scheme. 1.

3. Results

respectively (Fig. 1). PNA and Alcian blue staining intensities decreased with increasing concentrations of rottlerin. In particular, when cells were exposed to concentration of 1000 nM rottlerin, chondrogenic differentiation was completely blocked as demonstrated by the absence of Alcian blue staining. Chondrogenic differentiation is known to be regulated at 3 stages: cell proliferation, precartilage condensation and cartilage nodule formation [26,27]. Condensation could result from either an increase in cell proliferation or active cell movement that causes an increase in cell density without an increase in cell proliferation [3,9,10]. We first examined if rottlerin-suppressed chondrogenesis by regulating cell proliferation. Although chondrogenesis was significantly inhibited as shown in Fig. 1, exposure of cells to rottlerin did not affect apoptosis of chondrogenic competent cells as determined by direct cell counting (Fig. 2A) and FACS analysis (Fig. 2B). To examine the possible involvement of rottlerin in the control of cell motility, a wound-healing assay was performed to monitor the effects of rottlerin on the motility of chick wing bud mesenchymal cells. A rottlerin concentration as low as 250 nM nearly completely inhibited cell motility (Fig. 3A). Because prechondrogenic condensation begins with the aggregation of mesenchymal cells via migration and proliferation [2–4], suppression of cell migration by rottlerin inhibited precartilage condensation as demonstrated by PNA staining. Chondrogenic differentiation was also decreased by rottlerin treatment (Fig. 3B).

A

60 Con

Total cell number (x104 cells)

2.5. Cell migration assay

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Rotterlin

40 30 20 10 0 1

B Apoptotic cells (%)

626

90

pc

80

Con

70

Rotterlin

2

3 (day)

60 50 40 30 20 10

3.1. Effect of rottlerin on the migration of chick wing bud mesenchymal cells To investigate the role of rottlerin in chondrogenic differentiation, chick wing bud mesenchymal cells were treated with varying concentrations of rottlerin. Precartilage condensation and chondrogenic differentiation were analyzed by PNA and Alcian blue staining,

0 1

2

3 (day)

Fig. 2. The effect of rottlerin on wing mesenchymal cell migration is independent of apoptotic cell death. Wing mesenchymal cells were cultured at a density of 2  107 cells/ml and treated with rottlerin. (A) The total cell number was counted. (B) The percentages of apoptotic cells were quantified by flow cytometric analysis at the indicated days. Pc: positive control.

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Fig. 3. Effect of rottlerin on chick wing bud mesenchymal cell migration. Cell migration was accessed with wound-healing assays as described in Materials and methods. (A) Concentration-dependent study. Cultures were wounded with a micro-pipette tip and treated with different concentrations of rottlerin (0–1000 nM), a PKCd-selective inhibitor. Photos were taken at 15 h after wounding. (B) Wounded cultures were treated with 1000 nM rottlerin, photographed at 15 h after wounding, and stained with PNA at 3 days and Alcian blue at 5 days after seeding. Dotted lines delimit the initially wounded regions. Three independent experiments were performed for wound-healing assays (representative images are shown).

3.2. Integrin b1 and Src family kinases play a role in rottlerin-suppressed cell migration Cell migration is a complex process involving receptor-mediated adhesion, membrane protrusion and the formation of discrete cell–matrix adhesion sites linked to a reorganizing actin cytoskeleton [28–30]. Three structural elements, including an ECM ligand, its cell surface receptor and the cell cytoskeleton, are required for cell migration. Recent studies have shown that members of the integrin family, including the a5b1 fibronectin (FN) receptor, a2b1 and a3b1, and the vitronectin receptor avb3 interact reversibly with both ECM proteins and cytoskeletal structures [31]. Therefore, cell migration on ECM proteins could be regulated not only by the type of receptor/ligand interactions, but also by the ligand density on the substrate and the ligand affinity for cell adhesion receptors. First, possible rottlerin-induced alterations of FN and integrin b1 expressions in chick wing bud mesenchymal cells were examined by immunoblotting (Fig. 4A). The expression level of FN remained unchanged by rottlerin treatment. However, integrin b1 expression in chick wing bud mesenchymal cells was completely abolished. The phosphorylation levels of Src ((p)Src) and FAK were also decreased by rottlerin. To establish a role for integrin b1 on the motility of these cells, we performed a perturbation experiment using blocking antibodies

against b1 integrin. The functional blocking of integrin b1 suppressed the migration of wing bud mesenchymal cells, which supported a relationship between integrin b1 and rottlerinmodulated cell migration (Fig. 4B). Furthermore, the engagement of integrins to the ECM triggers the activation of protein tyrosine kinases, such as Src family and focal adhesion kinase (FAK) [32,33]. Next, the role of FAK in promoting cell motility was examined using siRNA. The siRNA against FAK partially suppressed the expression level of endogenous FAK in chick wing bud mesenchymal cells (data not shown). A wound-healing assay performed 24 h after electroporation of FAK specific siRNA showed that FAK knockdown suppressed cell migration (Fig. 4C) confirming a role for FAK in promoting cell migration. An inhibitor of Src family kinases, PP2, also inhibited the motility of cells. These observations suggest that Src family kinases play a role in integrin signaling in focal adhesion complexes during cell migration. 3.3. The effect of rottlerin on migration is independent of PKCd in chick limb bud mesenchymal cells Next, we asked the signaling mechanism involved in rottlerinregulated cell migration. Rottlerin is known as a selective inhibitor of PKCd. However, immunoblotting analysis confirmed that rottlerin did not inhibit PKCd (Fig. 5A). And inhibition of PKCd by

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Fig. 4. Integrin b1 and Src family kinases play roles in rottlerin-modulated cell migration. (A) Changes in the levels of FN, integrin b4, (p)Src, (p)FAK925, (p)FAK862, (p)FAK576, (p)FAK397, and FAK in control and rottlerin-treated cultures were determined by Western blotting at the indicated time points (days). (B) Cells were incubated with 2 mg/ml anti-b1 integrin blocking antibody and cultures were wounded with a micro-pipette tip and photographed at 15 h after wounding. (C) Cells were either electroporated with siRNA against FAK (siFAK) or treated with 10 mM PP2. Cultures were wounded with a micro-pipette tip and photographed at 15 h after wounding. Three independent experiments were performed for wound-healing assays (representative images are shown).

general PKC inhibitors such as Go¨6976 and Go¨6983 and Bisindolylmaleimide, did not affect the migration of chick wing bud mesenchymal cells in the wound-healing assay (Fig. 5B). These results strongly suggest that rottlerin suppresses cell migration independently of PKCd inhibition.

Our previous reports have shown that ERK and p38MAPK regulate mesenchymal cell chondrogenesis by modulating the expressions of cell adhesion molecules, including N-cadherin, fibronectin and integrin a5/b1 [34], and that Akt signaling is responsible for reorganizing the actin cytoskeleton to a cortical

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Fig. 5. Rottlerin-suppressed cell migration is independent of PKCd. (A) Changes in the levels of PKCa and PKCd in control and rottlerin-treated cultures were determined by Western blotting at the indicated time points (days). (B) Cell cultures were treated with 10 mM Go¨6976, 10 mM Go¨6983, or 10 mM Bisindolylmaleimide and were wounded with a micro-pipette tip and photographed at 15 h after wounding. Three independent experiments were performed for wound-healing assays and the representative images were presented.

pattern with a concomitant rounding of cells via activation of MMP2 [35]. Therefore, we asked if rottlerin affected the phosphorylation of these signaling molecules. Exposure to rottlerin suppressed the activities of chondro-positive signaling, p38MAPK and Akt, and stimulated the activity of a chondro-negative signal, Erk, in chick limb bud mesenchymal cells (Fig. 6A). However, modulation of p38MAPK or Erk signaling using specific inhibitors did not affect the migration of chick wing bud mesenchymal cells in the woundhealing assay. In contrast, Akt inhibition dramatically reduced the migration of chick wing bud mesenchymal cells in the woundhealing assay (Fig. 6B) and integrin b1 expression and phosphorylation of Src as analyzed by Western blotting (Fig. 6C). These results suggest that rottlerin suppresses cell migration via inhibition of Akt activity, independent of PKCd inhibition. 4. Discussion Although it is known that condensation during cartilage formation depends on cell proliferation in response to apical ectodermal ridge (AER) signals, it is also facilitated by the directed distal migration of subridge mesenchymal cells toward the AER, which requires a coordinated regulation between cell adhesion, cell contractility and actin cytoskeletal remodeling [36,37]. The mechanisms involved with cell migration have not been well studied. Recent reports show that rottlerin used in this study is involved in the apoptotic cell death and migration of several myeloma cells [14–17]. Consistent with this, we also found the involvement of rottlerin in migration of chondrogenic competent cells. Therefore, the investigation of this migratory mechanism by rottlerin could provide the pivotal information for the better understating regarding cellular migratory and condensation mechanism during chondrogenic differentiation of chick limb bud

mesenchymal cells. Here, we demonstrate for the first time the possibility that rottlerin-modulated Akt signaling is involved in the migratory regulation of chondrogenic progenitors via modulation of integrin b1-mediated focal adhesion activity. Previously, our laboratory demonstrated that Akt signaling induced the reorganization of the actin cytoskeleton into cortical pattern with parallel rounding of cells, a typical characteristic of differentiated chondrogenic competent cells [38]. This study could provide a linkage among Akt, focal adhesion activity, and actin cytoskeleton reorganization. Cell motility results from a highly orchestrated set of cell migratory processes that are mediated by integrins in particular. Integrins are receptor proteins that form focal adhesion complexes that connect the ECM with the actin cytoskeleton. Integrin complexes both transmit signals into the cell interior and receive signals from within the cells. In this study, cell exposure to rottlerin blocked integrin b1 expression at focal adhesion complexes and suppressed migration of chick limb bud mesenchymal cells. Integrin engagement stimulates the activity of numerous other signaling molecules, including protein tyrosine kinases (PTKs), such as Src and FAK [39], tyrosine phosphatases [40], cAMP-dependent protein kinase and protein kinase C (PKC) [41]. Focal adhesion kinase (FAK) is a non-receptor associated tyrosine kinase that is localized at focal adhesion sites. The amino-terminal domain of FAK can bind with the cytoplasmic domain of b-integrin and growth factor receptors [42]. Integrin binding has been shown to induce FAK autophosphorylation following the activation of a number of Src homology 2 (SH2) containing proteins, including Src, and results in dramatic effects on the actin cytoskeleton, cell migration and cell proliferation [43]. In this study, we found that FAK is a central signal from the ECM to cSrc family tyrosine kinase in rottlerin-modulated cell migration.

Fig. 6. Rottlerin suppresses cell migration via modulation of Akt activity. (A) Changes in the levels of (p)Akt, pp38MAPK, and (p)Erk in control and rottlerin-treated cultures were determined by Western blotting at the indicated time points (days). (B) Cell cultures were treated with 10 mM PD98059, 10 mM SB203580 and 500 nM Akt inhibitor IV and were wounded with a micro-pipette tip and photographed at 15 h after wounding. (C) Changes in the levels of integrin b4 and (p)Src in control and Akt inhibitor-treated cultures were determined by Western blotting at the indicated time points (days).

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For adherent cells, integrins are clustered at the cell attachment sites in focal adhesions. At these sites, integrins not only anchor cells to the ECM, but also integrate the attachment of the exterior of the cells with the reorganizing intracellular actin cytoskeleton. The association of integrin b1 with an actin filament bundle is an intriguing possibility that might relate to the mechanism of chick limb bud mesenchymal cell migration found in this study. Therefore, additional studies on the involvement of this actin bundle in cell migration should be enlightening. In summary, our results suggest that exogenous rottlerin may act as a negative regulator for cell migration, which is an essential step for prechondrogenic condensation, through regulation of integrin b1 signaling at focal adhesion complex via the modulation of Akt activity.

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Acknowledgements

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This research was supported by a Korea Research Foundation (KRF) grant funded by the Korea Government (C00731) to E.J. Jin and supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (R01-2006-00010756-0) to S.S. Kang.

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