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Synchronous Activation of ERK and Phosphatidylinositol 3-Kinase Pathways Is Required for Collagen and Extracellular Matrix Production in Keloids*

  • Ivor J. Lim
    Footnotes
    Affiliations
    Department of Surgery, National University of Singapore, 5, Lower Kent Ridge Road, Singapore 119074
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  • Toan-Thang Phan
    Footnotes
    Affiliations
    Department of Surgery, National University of Singapore, 5, Lower Kent Ridge Road, Singapore 119074
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  • Ee-Kim Tan
    Affiliations
    Department of Surgery, National University of Singapore, 5, Lower Kent Ridge Road, Singapore 119074

    Department of Orthopaedic Surgery, National University of Singapore, 5, Lower Kent Ridge Road, Singapore 119074
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  • Thi-Thanh T. Nguyen
    Affiliations
    Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre, Singapore 169610
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  • Evelyne Tran
    Affiliations
    Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre, Singapore 169610
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  • Michael T. Longaker
    Affiliations
    Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148
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  • Colin Song
    Affiliations
    Department of Plastic Surgery/Burns Centre, Singapore General Hospital, Outram Road, Singapore 169608
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  • Seng-Teik Lee
    Affiliations
    Department of Plastic Surgery/Burns Centre, Singapore General Hospital, Outram Road, Singapore 169608
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  • Hung-The Huynh
    Correspondence
    To whom correspondence should be addressed. Tel.: 65-436-8347; Fax: 65-226-5694
    Affiliations
    Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre, Singapore 169610
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  • Author Footnotes
    * This work was supported by SingHealth Grant EX008/2001 and National Medical Research Council of Singapore Grant NMRC/0541/2001 (to H.-T. H.) and by Biomedical Research Council of Singapore Grant 02/1/21/19/106 (to I. J. L. and T.-T. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Both authors contributed equally to this work.
Open AccessPublished:August 07, 2003DOI:https://doi.org/10.1074/jbc.M305759200
      Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2–5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I–III, laminin β2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I–III production and significantly decreased laminin β2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I–III and laminin β2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.
      Keloids are fibroproliferative scars that affect only humans (
      • Rockwell W.B.
      • Cohen I.K.
      • Ehrlich H.P.
      ,
      • Niessen F.B.
      • Spauwen P.H.
      • Schalkwijk J.
      • Kon M.
      ). As opposed to hypertrophic scarring, which might result from transient exuberant remodeling of scar collagen and the extracellular matrix (ECM)
      The abbreviations used are: ECM, extracellular matrix; KK, keloid keratinocyte; KF, keloid fibroblast; NF, normal fibroblast; IGF, insulin-like growth factor; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; JNK, c-Jun N-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
      1The abbreviations used are: ECM, extracellular matrix; KK, keloid keratinocyte; KF, keloid fibroblast; NF, normal fibroblast; IGF, insulin-like growth factor; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; JNK, c-Jun N-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
      in response to skin stress and strain (
      • Ehrlich H.P.
      • Desmouliere A.
      • Diegelmann R.F.
      • Cohen I.K.
      • Compton C.C.
      • Garner W.L.
      • Kapanci Y.
      • Gabbiani G.
      ), keloids result from a pathological wound healing response. The keloid scar is well known clinically for its unique semiautonomous nature, with lesions that “overflow” the original wound margins and recur after excision. That they are notoriously resistant to therapy can be seen by the numerous treatment modalities available, none of which can be said to be truly effective (
      • Niessen F.B.
      • Spauwen P.H.
      • Schalkwijk J.
      • Kon M.
      ,
      • Urioste S.S.
      • Arndt K.A.
      • Dover J.S.
      ).
      The fibroblast, which is responsible for collagen-ECM production (
      • Cohen I.K.
      • Peacock Jr., E.E.
      ), has traditionally been the subject of study in keloid pathogenesis. It is, however, under continuous paracrine signal bombardment from neighboring cells, which influences its behavior and vice versa. This phenomenon was first studied in normal skin (
      • Mackenzie I.C.
      ,
      • Fusenig N.E.
      ), and it is now acknowledged that the actively secreting keratinocyte (
      • Boyce S.T.
      ,
      • Katz A.B.
      • Taichman L.B.
      ) has a major role to play in normal skin differentiation and hemostasis (termed “epithelial-mesenchymal interactions”). Finally, in contributing to the general endocrine milieu, keratinocytes ultimately affect systemic homeostasis (
      • Maas-Szabowski N.
      • Shimotoyodome A.
      • Fusenig N.E.
      ,
      • Xue H.
      • McCauley R.L.
      • Zhang W.
      ,
      • Garner W.L.
      ).
      Epithelial-mesenchymal interactions were recently extrapolated to keloid pathogenesis following the clinical observation that the initiating event for keloid formation is invariably skin trauma, ranging from the insignificant to the severe (
      • Lim I.J.
      • Phan T.-T.
      • Bay B.H.
      • Qi R.
      • Huynh H.-T.
      • Tan W.T.
      • Lee S.-T.
      • Longaker M.T.
      ,
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ,
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ). The secretory role of the intrinsically abnormal keloid keratinocyte (KK) undergoes dramatic change following such injury to stimulate local and systemic responses. Unlike normal skin, these KK-derived regulatory paracrine factors are secreted either abnormally or in altered proportions. The KK may itself be insensitive to paracrine feedback from the fibroblast. Taken together, these data strongly suggest that fibroblast behavior in terms of cell proliferation and collagen-ECM synthesis is dictated to a significant degree by the overlying keratinocyte. In the case of keloids, both keloid fibroblasts (KFs) and KKs appear to have underlying abnormalities in the paracrine feedback loop, stimulating fibroproliferative mechanisms and tilting the balance of collagen-ECM synthesis toward excess deposition and accumulation. A genetic predisposition to this aberrant cell response remains incompletely understood (
      • Niessen F.B.
      • Spauwen P.H.
      • Schalkwijk J.
      • Kon M.
      ,
      • Omo-Dare P.
      ,
      • Bayat A.
      • Bock O.
      • Mrowietz U.
      • Ollier W.E.
      • Ferguson M.W.
      ,
      • Marneros A.G.
      • Norris J.E.
      • Olsen B.R.
      • Reichenberger E.
      ).
      A previous study by our group demonstrated increased normal fibroblast (NF) and KF cell proliferation in defined serum-free medium co-culture with KKs compared with that in co-culture with normal keratinocytes, the latter of which was significantly higher than NF or KF cell proliferation in single cell culture (
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ). It was subsequently demonstrated that NFs and KFs exposed to KK paracrine secretions in co-culture secreted increased amounts of collagens I and III, which were again higher than in NF or KF single cell culture. The transmission electron microscopic morphology of collagen-ECM of NFs co-cultured with KKs approaches that of in vivo keloid tissue sections (
      • Lim I.J.
      • Phan T.-T.
      • Bay B.H.
      • Qi R.
      • Huynh H.-T.
      • Tan W.T.
      • Lee S.-T.
      • Longaker M.T.
      ). More recent work from our group investigating the role of the insulin-like growth factor (IGF) system of mitogens in keloids demonstrated heightened activation of ERK and PI3K pathways in KFs co-cultured with KKs, resulting in increased KF proliferation (
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ). This activation was again greatly enhanced in co-culture compared with single cell culture. In that study, IGF-1 interaction with the IGF-1 receptor appeared to be modulated by the bioavailability of IGF-binding protein-3 (
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ).
      From the above studies, it became clear that fibroproliferation and collagen-ECM production are closely related. We hypothesized that keloid collagen and ECM component production is also linked to a MAPK cascade, specifically the MEK-ERK and PI3K pathways. To test this hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2–5 days. KF cell lysate was subjected to Western analysis to assess the activity of these two pathways and their downstream components affecting cell transcription. The conditioned medium from the co-culture was assayed to investigate fibroblast production of collagens I–III, which form the main structural fibers in skin, as well as laminin β2 and fibronectin, which are linking glycoproteins that have roles in cell adhesion and differentiation and which are found predominantly in the basal lamina, in the presence or absence of inhibitors of MEK1/2 (U0126) and PI3K (LY294002). Additionally, the activities of the stress-related MAPK pathways JNK and p38 kinase were also investigated using the p38 kinase inhibitor SB203580 and the JNK inhibitor SP600125 to determine their role in keloid collagen and ECM component production.

      EXPERIMENTAL PROCEDURES

      Reagents

      U0126, LY294002, rabbit anti-phospho-Ser217/Ser221 MEK1/2, rabbit anti-phospho-Ser473 Akt, mouse anti-phospho-Thr202/Tyr204 ERK1/2, rabbit anti-Akt, and mouse anti-ERK antibodies were purchased from New England Biolabs Inc. (Beverly, MA). SB203580 and SP600125 were supplied by Calbiochem-Novabiochem. Mouse anti-collagen I–III antibody was from Monosan (Am Uden, The Netherlands). Mouse antilaminin β2 and mouse anti-fibronectin antibodies were supplied by Transduction Laboratories (Lexington, KY). Mouse anti-α-tubulin, rabbit anti-p38 kinase, rabbit anti-phospho-Tyr182 p38 kinase, rabbit anti-JNK2, and rabbit anti-phospho-Thr183/Tyr185 JNK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit secondary antibodies were purchased from Pierce. The chemiluminescence detection system (ECL) was supplied by Amersham Biosciences. Tissue culture Petri dishes, 6-well plates, and 96-well plates were purchased from Nunc (Naperville, IL). Medium, fetal bovine serum, and penicillin/streptomycin were from Invitrogen.
      U0126, LY294002, SB203580, and SP600125 are MEK1/2-specific, nonselective PI3K, p38 kinase-specific, and JNK-specific inhibitors, respectively. They were dissolved in dimethyl sulfoxide (with the final concentration never exceeding 0.1%) and stored frozen under light-protected conditions at –20 °C.

      Earlobe KKs and Fibroblast Data Base

      Four strains of keloid-derived fibroblasts (KF16, KF17, KF18, and KF48) and two strains of keloid-derived keratinocytes (KK18 and KK48) were obtained from excised earlobe keloid specimens. No patient had previously received any treatment for the keloids. A full history and clinical examination were supplemented with color slide photo documentation. Written informed consent was obtained before operative excision, and a portion of all excised specimens was sent to the Singapore General Hospital Department of Pathology for histological confirmation of keloid identity.

      Cell Culture

      Keratinocyte Culture with Earlobe Keloids—Passage 2 keratinocytes were obtained from excised specimens as previously described (
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ). Briefly, excised earlobe keloid specimens were washed with Hanks' balanced salt solution containing 150 μg/ml gentamycin and 7.5 μg/ml Fungizone followed by plain phosphate-buffered saline until the solution became clear. The specimens were then cut into 5 × 10-mm pieces, and the epidermis was scored. Dispase (5 mg/ml) in Hanks' balanced salt solution was added, and the specimens were left overnight at 4 °C. The epidermis was carefully scraped off with a scalpel the next day and incubated in a solution of 0.25% trypsin, 0.1% glucose, and 0.02% EDTA for 10 min. Trypsin action was quenched by Dulbecco's modified Eagle's medium and 10% fetal bovine serum upon intercellular separation. Suspended cells were transferred to tubes and centrifuged at 1000 rpm for 8 min. The cells were then isolated and seeded in keratinocyte culture medium (80 ml of Dulbecco's modified Eagle's medium supplemented with 20 ml of fetal bovine serum, 10 ng/ml epidermal growth factor, 1 × 10–9m cholera toxin, and 0.4 μg/ml hydrocortisone) at 1 × 105 cells/cm2 for 24 h before transfer to keratinocyte growth medium (Clonetics Corp.). Cell strains were maintained and stored at –150 °C until used. Only cells from the second passage were used in all experiments.
      Fibroblast Culture with Earlobe Keloids—Remnant keloid dermis was minced and incubated in a solution of collagenase I (0.5 mg/ml) and trypsin (0.2 mg/ml) for 6 h at 37 °C. Cells were pelleted and grown in tissue culture flasks. Cell strains were maintained and stored at –150 °C until used. Only cells from the second passage were used in all experiments.
      Keratinocyte-Fibroblast Co-culture—Keratinocytes from samples KK18 and KK48 were thawed, centrifuged, and recounted. Cells were seeded at a density of 4 × 105 cells/cm2 on Transwell clear polyester membrane inserts (0.4-μm pore size, 0.3-cm2 area; Corning-Costar). Cells were maintained for 4 days in serum-free keratinocyte growth medium until 100% confluent in monolayer. The medium was then changed to serum-free defined fibroblast growth medium, and the cells were raised to air-liquid interface for another 3 days, allowing keratinocytes to stratify and to reach terminal differentiation (
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ).
      Fibroblasts from samples KF16, KF17, KF18, and KF48 were thawed and seeded on 24-well plates at a density of 5 × 104 cells/well in defined fibroblast growth medium for 3–4 days until 100% confluent. Both the cultured keratinocytes on membrane inserts and the cultured fibroblasts on plates were washed twice with phosphate-buffered saline to remove the old medium before placing the inserts on the plates, commencing KK-KF co-culture in fresh serum-free defined fibroblast growth medium with or without varying concentrations of U0126, LY294002, SB203580, or SP600125 for 2 or 5 days. At day 2 or 5, membrane inserts with the cultured keratinocytes were removed, and the conditioned medium was collected for collagen, laminin, and fibronectin analysis. Fibroblasts were also harvested for Western blot analysis.

      Western Blotting

      To detect collagens, fibronectin, and laminin in the conditioned medium, 1 ml of conditioned medium was concentrated using a Centricon centrifuge (Millipore Corp., Bedford, Mass.) and then separated by SDS-PAGE under reducing conditions and electroblotted onto nitrocellulose membrane. Blots were incubated with the indicated antibody and horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (1:7500 dilution). Blots were visualized with the ECL chemiluminescence detection system as recommended by the manufacturer.
      To examine the effects of U0126 on the expression of ERK1/2, phospho-Ser217/Ser221 MEK1/2, and phospho-Thr202/Tyr204 ERK1/2; LY294002 on the expression of Akt and phospho-Ser473 Akt; SB203580 on the expression of p38 kinase and phospho-Tyr182 p38 kinase; and SP600125 on the expression of JNK and phospho-Thr183/Tyr185 JNK, KF cells plated at a density of 5 × 104 cells were co-cultured with KKs for the indicated times (2 or 5 days) in defined fibroblast growth medium in the presence or absence of the indicated concentrations of inhibitors. Following this treatment, KF cells were lysed in lysis buffer (1 mm CaCl2, 1 mm MgCl2, 1% Nonidet P-40, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μm phenylmethylsulfonyl fluoride, and 100 μm NaVO4), and Western blot analysis was performed. Blots were incubated with the indicated antibodies, and horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (1:7500 dilution). All of the primary antibodies were used at a final concentration of 1 μg/ml. Blots were then visualized with the ECL chemiluminescence detection system as recommended by the manufacturer.

      Cell Proliferation Assay

      The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay that tests the metabolic activity of viable cells and is used for indirect cell quantification (
      • Hansen M.B.
      • Nielsen S.E.
      • Berg K.
      ,
      • Mosmann T.
      ). This technique has been used by our group in two previous studies (
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ,
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ) and was used in this study to assess the fibroproliferative response under co-culture conditions to the MEK1/2 inhibitor U0126 and the PI3K inhibitor LY294002.

      Statistical Analysis

      For quantitative analysis, the sum of the density of bands corresponding to protein blotting with the antibody under study was calculated, and the amount of α-tubulin was normalized. Differences in cell number and the levels of proteins under studied were analyzed by the Kruskal-Wallis test.

      RESULTS

      Co-culture of NFs or KFs with KKs Results in Increased Fibroproliferation and Collagen I–III Production—As a confirmatory study on the basal proliferative behavior and collagen production characteristics of three of the KF test samples (KF16, KF17, and KF18), a 5-day co-culture with KKs (KK18) was performed. This was followed by cell proliferation assessment using the MTT assay (Fig. 1A) and Western blotting of the conditioned medium to assess collagen I–III production (Fig. 1B). KFs demonstrated increased cell proliferation and collagen I–III production in co-culture with KKs, and the levels were significantly higher than those obtained with NFs cocultured with KKs. These results correlate with those of our previous studies (
      • Lim I.J.
      • Phan T.-T.
      • Bay B.H.
      • Qi R.
      • Huynh H.-T.
      • Tan W.T.
      • Lee S.-T.
      • Longaker M.T.
      ,
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ), establishing their expected behavior.
      Figure thumbnail gr1
      Fig. 1Effects of KK18 on KF16, KF17, and KF18 proliferation and collagen I–III secretion. A, NFs and the KF16, KF17, and KF18 cell strains were co-cultured with KK18 cells for 5 days as described under “Experimental Procedures.” Cell proliferation of the study groups at day 5 of co-culture was determined using the MTT assay. Bars with different letters are significantly different from one another (p < 0.01). Data are expressed as the mean ± S.E. of six samples. Results shown are representative of three independent experiments. B, for analysis of collagen secretion, the conditioned medium was harvested on day 5. One ml of conditioned medium was concentrated 10 times and subjected to Western blot analysis as described under “Experimental Procedures.” Blots were incubated with mouse anti-collagen I–III antibody.
      Co-culture of NFs or KFs with KKs Results in Increased Activation of the PI3K and MEK-ERK Pathways—A confirmatory study on the activity of both the PI3K and MEK-ERK pathways of three of the KF test samples (KF16, KF17, and KF18) in 5-day co-culture with KKs (KK18) was also performed. KFs co-cultured with KKs demonstrated increased phosphorylation of Akt-1 downstream of the PI3K pathway (Fig. 2C), as well as increased phosphorylation of MEK1/2 (Fig. 2D), ERK1/2 (Fig. 2E), and Elk-1 (Fig. 2G), again consistent with our previous study (
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ).
      Figure thumbnail gr2
      Fig. 2Effects of KK18 cells on activation of MEK1/2, ERK, Akt, and Elk-1 in KF16, KF17, and KF18 cells. NFs and the KF16, KF17, and KF18 cell strains were co-cultured with KK18 cells for 5 days as described under “Experimental Procedures.” For analysis of activated ERK and Akt-1, cells were harvested at day 5 and lysed for Western blot analysis as described under “Experimental Procedures.” Blots were incubated with mouse anti-α-tubulin (A), rabbit anti-Akt-1 (B), rabbit anti-phospho (p)-Ser473 Akt (C), rabbit anti-phospho-Ser217/Ser221 MEK1/2 (D), mouse anti-ERK (E), mouse anti-phospho-Thr202/Tyr204 ERK1/2 (F), and mouse anti-phospho-Ser383 Elk-1 (G) antibodies. Experiments were repeated three times with similar results.
      Addition of the MEK1/2-specific Inhibitor U0126 Decreases KF Proliferation in Co-culture with KKs—The proliferative characteristics of KF18 in co-culture with KK18 over a period of 5 days in the presence or absence of the MEK1/2-specific inhibitor U0126 (10 μm) were assessed using the MTT assay as described above. In the absence of U0126, KF18 cell proliferation was progressive, whereas the addition of U0126 resulted in progressively decreasing cell proliferation (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Effects of U0126 on KK18-induced KF18 cell proliferation. KK18 cells were co-cultured with KF18 cells in the presence or absence of 10 μm U0126 as described under “Experimental Procedures.” KF18 cell proliferation was determined daily for 5 days using the MTT assay. Bars with different letters are significantly different from one another (p < 0.01). Data are expressed as the mean ± S.E. of six samples. Results shown are representative of three independent experiments.
      Increasing Concentrations of U0126 Progressively Decrease Cell Proliferation and ERK Activation—KF18 was co-cultured with KK18 for 2 days in the absence or presence of different concentrations of U0126 (2, 4, 6, and 8 μm). A 2-day co-culture was selected, as the proliferation of KFs was seen to be progressive with time as long as they were co-cultured with KKs, without the need for a 5-day co-culture. KF18 cell proliferation was noted to be progressively reduced by increasing concentrations of U0126, especially between 2 and 6 μm (p < 0.01) (Fig. 4A). Increasing concentrations of U0126 also progressively decreased ERK1/2 phosphorylation, which was synchronous with the falling KF proliferation rates above (Fig. 4C).
      Figure thumbnail gr4
      Fig. 4Effects of U0126 on KK18-induced KF18 proliferation and activation of ERK. KK18 cells were co-cultured with KF18 cells in the absence or presence of the indicated concentrations of U0126 for 2 days as described under “Experimental Procedures.” Cell proliferation of the study groups at day 2 of co-culture was determined using the MTT assay (A). Bars with different letters are significantly different from one another (p < 0.01). Data are expressed as the mean ± S.E. of six samples. Results shown are representative of three independent experiments. For analysis of activated ERK1/2, cells were harvested at day 2 and lysed for Western blot analysis as described under “Experimental Procedures.” Blots were incubated with mouse anti-ERK (B) and mouse anti-phospho (p)-Thr202/Tyr204 ERK1/2 (C) antibodies. Experiments were repeated three times with similar results.
      Increasing Concentrations of U0126 Decrease Fibronectin Production and Abrogate Collagen I–III and Laminin β2 Production—A 2-day co-culture of KF18 with KK18 in the presence or absence of increasing concentrations of the MEK1/2-specific inhibitor U0126 resulted in complete abrogation of collagen I–III production at 4 μm and above (Fig. 5A). Complete nullification of laminin β2 production was also seen upon the addition of U0126 at 2 μm and above (Fig. 5B). Fibronectin production was decreased by increasing concentrations of U0126, but was never completely abrogated. Of note were the different in-gel migration characteristics, which reflected a change in the molecular mass of the molecule (Fig. 5C). We speculate this to be due to a change in side chain moiety attachment, which might have been altered in the presence of MEK inhibition.
      Figure thumbnail gr5
      Fig. 5Effects of U0126 on KK18-induced collagen, laminin β2, and fibronectin secretion by KF18 cells. KK18 cells were co-cultured with KF18 cells in the absence or presence of the indicated concentrations of U0126 for 2 days as described under “Experimental Procedures.” The conditioned medium was collected on day 2, and 1 ml of conditioned medium was concentrated 10 times and subjected to Western blotting as described under “Experimental Procedures.” Blots were incubated with mouse anti-collagen I–III (A), mouse anti-laminin β2 (B), and mouse anti-fibronectin (C) antibodies.
      Increasing Concentrations of LY294002 Progressively Decrease Cell Proliferation and Akt-1 Activation—KF18 was cocultured with KK18 for 2 days in the absence or presence of different concentrations of LY294002 (2, 4, 6, and 8 μm). Similar to the results obtained with U0126 inhibition, KF18 cell proliferation was noted to be progressively reduced by increasing concentrations of LY294002, with a significant difference seen especially between 2 and 4 μm (p < 0.01) (Fig. 6A). Increasing concentrations of LY294002 progressively decreased Akt-1 phosphorylation (Fig. 6C) and abruptly so beyond concentrations of 4 μm. Akt-1 production by itself was unchanged by the treatment (Fig. 4B).
      Figure thumbnail gr6
      Fig. 6Effects of LY294002 on KK18-induced KF18 proliferation and activation of Akt-1. KK18 cells were co-cultured with KF18 cells in the absence or presence of the indicated concentrations of LY294002 for 2 days as described under “Experimental Procedures.” Cell proliferation of the study groups at day 2 of co-culture was determined using the MTT assay (A). Bars with different letters are significantly different from one another (p < 0.01). Data are expressed as the mean ± S.E. of six samples. Results shown are representative of three independent experiments. For analysis of activated Akt-1, cells were harvested at day 2 and lysed for Western blot analysis as described under “Experimental Procedures.” Blots were incubated with rabbit anti-Akt-1 (B) and rabbit anti-phospho (p)-Ser473 Akt-1 (C) antibodies. Experiments were repeated three times with similar results.
      Increasing Concentrations of LY294002 Abrogate Collagen I–III Production and Decrease Laminin β2 and Fibronectin Production—KF18 was co-cultured with KK18 for 2 days in the presence or absence of increasing concentrations of the PI3K-specific inhibitor LY294002. Complete abrogation of collagen I–III production was seen at concentrations of 4 μm and above (Fig. 7A). Laminin β2 production was substantially reduced upon the addition of LY294002 at 2 μm and above, but was not completely nullified (Fig. 7B). Similar to the situation of U0126 inhibition of ERK phosphorylation seen above, a decrease in fibronectin production associated with a change in the migration speed was seen in this instance, but fibronectin production was not abrogated (Fig. 7C).
      Figure thumbnail gr7
      Fig. 7Effects of LY294002 on KK18-induced collagen, laminin β2, and fibronectin secretion by KF18 cells. KK18 cells were co-cultured with KF18 cells in the absence or presence of the indicated concentrations of LY294002 for 2 days as described under “Experimental Procedures.” The conditioned medium was collected on day 2, and 1 ml of conditioned medium was concentrated 10 times and subjected to Western blotting as described under “Experimental Procedures.” Blots were incubated with mouse anti-collagen I–III (A), mouse anti-laminin β2 (B), and mouse anti-fibronectin (C) antibodies.
      U0126 Inhibition of MEK1/2 Is as Efficacious as LY294002 Inhibition of PI3K in Decreasing KF Proliferation and Abolishing Collagen-ECM Production in Co-culture with KKs—As a side-by-side comparison study, a 2-day co-culture of KF18 with KK18 was performed in the absence or presence 8 μm U0126 or LY294002, with cell proliferation assayed using MTT. The concentration of 8 μm was selected for maximal inhibition of KF proliferation and collagen-ECM production based on the data above. The rate of cell proliferation of KF18 in co-culture with KK18 was suppressed in the presence of either inhibitor, with no statistical difference compared with KF18 in single cell culture. This was markedly diminished compared with KK18-KF18 co-culture in the absence of either inhibitor (Fig. 8A). A concurrent assay of KF18 cell lysate showed clear suppression of ERK1/2 and Akt-1 phosphorylation by their respective inhibitors in this comparison (Fig. 8, B–E). The conditioned media from the co-culture groups were assayed for collagens I–III, laminin β2, and fibronectin. Concurrent with earlier findings, collagen I–III and laminin β2 production was totally abolished in the presence of either U0126 or LY294002 (data not shown). Fibronectin production was diminished, but not totally abrogated; and in-gel migration speeds were diminished (data not shown). These data reinforce the suggestion that cross-talk between the MEK-ERK and PI3K pathways exists and that mutual activation of these two pathways is required for ECM secretion.
      Figure thumbnail gr8
      Fig. 8Effects of U0126 and LY294002 on KK18-induced KF18 proliferation and ERK and Akt-1 phosphorylation. KK18 cells were co-cultured with KF18 cells in the absence or presence of 8 μm U0126 or LY294002 for 48 h as described under “Experimental Procedures.” Cell proliferation of the study groups at day 2 of co-culture was determined using the MTT assay (A). Bars with different letters are significantly different from one another (p < 0.01). Data are expressed as the mean ± S.E. of six samples. Results shown are representative of three independent experiments. For analysis of activated ERK and Akt-1, cells were harvested at day 2 and lysed for Western blot analysis as described under “Experimental Procedures.” Blots were incubated with mouse anti-ERK (B), mouse anti-phospho (p)-Thr202/Tyr204 ERK1/2 (C), rabbit anti-Akt-1 (D), and rabbit anti-phospho-Ser473 Akt-1 (E) antibodies. Experiments were repeated three times with similar results.
      Addition of SB203580, a p38 Kinase-specific Inhibitor, Has No Effect on Collagen, Fibronectin, or Laminin β2 Production—To further examine the involvement of the stress-related MAPK cascade p38 kinase in collagen-ECM production, KF48 was co-cultured with KK48 for 2 days in the presence or absence of the p38 kinase-specific inhibitor SB203580 at 10 or 15 μm. SB203580 appeared to reduce the levels of phospho-p38 kinase, but did not completely inhibit p38 phosphorylation at Tyr182 (Fig. 9, E and F). No significant changes were observed in the secreted levels of collagen, laminin β2, or fibronectin compared with the situation of KF48 co-cultured with KK48 in the absence of this inhibitor (Fig. 10, A–C).
      Figure thumbnail gr9
      Fig. 9Effects of SB203580 and SP600125 on KK48-induced KF48 p38 kinase and JNK phosphorylation. KK48 cells were co-cultured with KF48 cells in the absence or presence of 10 or 15 μm SB203580 (the p38 kinase-specific inhibitor) or 20 or 50 μm SP600125 (the JNK-specific inhibitor) for 48 h as described under “Experimental Procedures.” For analysis of p38 kinase and JNK phosphorylation, cells were harvested at day 2 and lysed for Western blot analysis as described under “Experimental Procedures.” Blots were incubated with mouse anti-ERK (A), mouse anti-phospho-Thr202/Tyr204 ERK1/2 (B), rabbit anti-Akt-1 (C), rabbit anti-phospho-Ser473 Akt-1 (D), rabbit anti-p38 kinase (E), rabbit anti-phospho-Tyr182 p38 kinase (F), rabbit anti-JNK2 (G), and rabbit anti-phospho-Thr183/Tyr185 JNK (H) antibodies. Experiments were repeated three times with similar results.
      Figure thumbnail gr10
      Fig. 10Effects of SB203580 and SP600125 on KK48-induced collagen, laminin β2, and fibronectin secretion by KF48 cells. KK48 cells were co-cultured with KF48 cells in the absence or presence of the indicated concentrations of SB203580 and SP600125 for 2 days as described under “Experimental Procedures.” The conditioned medium was collected on day 2, and 1 ml of conditioned medium was concentrated 10 times and subjected to Western blotting as described under “Experimental Procedures.” Blots were incubated with mouse anti-collagen I–III (A), mouse anti-laminin β2 (B), and mouse anti-fibronectin (C) antibodies.
      Addition of SP600125, a JNK-specific Inhibitor, Has No Effect on Collagen, Fibronectin, or Laminin β2 Production—The role of the stress-related MAPK cascade JNK in collagen-ECM production was also investigated to complement the above study on the role of p38 kinase. To achieve this, KF48 was co-cultured with KK48 for 2 days in the presence or absence of the JNK-specific inhibitor SP600125 at 20 or 50 μm. SP600125 was seen to effectively reduce the levels of phospho-JNK (Fig. 9, G and H). Once again, no significant changes were observed in the secreted levels of collagen, laminin β2, or fibronectin compared with the situation of KF48 co-cultured with KK48 in the absence of this inhibitor (Fig. 10, A–C).

      DISCUSSION

      The hallmark of the keloid is aberrant, excess collagen-ECM deposition and accumulation, which are directly responsible for the gross appearance of this scar tissue. The ECM is a complex structural entity surrounding and supporting mammalian cells comprising collagen, multi-adhesive matrix proteins, proteoglycans, and hyaluronan. These ECM components have complex interactions not just with each other, but also with the cells that they envelop, in the form of cell adhesion, cell-cell signaling, tissue function, and wound repair (
      • Aumailley M.
      • Gayraud B.
      ). In the skin, the main structural proteins are collagens I and III, predominantly secreted by fibroblasts and to a small degree by epithelial cells. The laminins and fibronectin are linking glycoproteins that anchor keratinocyte and fibroblast surfaces to the basal lamina in close relationship to collagen IV, which forms the main structural protein for the basement membrane-basal lamina complex responsible for the adherence of the epidermis to the underlying dermis. In addition to cell adhesion, laminins and fibronectin are also important for cell migration and differentiation (
      • Aumailley M.
      • Smyth N.
      ).
      Collagen-ECM secretion in fibroblasts has been extensively investigated in osteoblasts, the specialized fibroblasts of bone, and appears to be regulated by a variety of cytokines, including transforming growth factor-β and bone morphogenetic protein-2, via signals through their interaction with specific serine/threonine kinase cell-surface receptors (
      • Centrella M.
      • Horowitz M.C.
      • Wozney J.M.
      • McCarthy T.L.
      ,
      • Noda M.
      • Rodan G.A.
      ), which also control osteoblast cell proliferation and differentiation in vitro. The α1(I) collagen gene appears to be up-regulated by downstream activation of MAPKs (specifically the ERK kinase-ERK pathway) via protein kinase C and phosphorylated tyrosine kinase pathways (
      • Palcy S.
      • Goltzman D.
      ). In terms of aberrant fibroproliferation, as seen in liver cirrhosis, acetaldehyde appears to activate the ERK and PI3K pathways via protein kinase C, resulting in the up-regulation of α2(I) collagen (
      • Svegliati-Baroni G.
      • Ridolfi F.
      • Di Sario A.
      • Saccomanno S.
      • Bendia E.
      • Benedetti A.
      • Greenwel P.
      ). MAPKs may also be activated by a mechanically coupled transcriptional circuit via intercellular integrins to induce the expression of filamin A in fibroblasts (
      • D'Addario M.
      • Arora P.D.
      • Ellen R.P.
      • McCulloch C.A.
      ). There are four other distinguishable MAPK modules in mammalian cells in addition to ERK that regulate cell growth and differentiation: the JNK and p38 kinase cascades, which are regulated by inflammatory cytokines and cellular stress, and the ERK3 and ERK5 cascades, which are related to serum stress and other as yet undefined stimuli (
      • Schaeffer H.J.
      • Weber M.J.
      ). At least one component of aberrant fibroproliferation in scleroderma has recently been found to be p38 kinase-dependent via transforming growth factor-β, regulating collagen I mRNA and α2(I) collagen promoter activity (
      • Sato M.
      • Shegogue D.
      • Gore E.A.
      • Smith E.A.
      • McDermott P.J.
      • Trojanowska M.
      ). The ERK and p38 kinase cascades, modulated by HSP27, additionally appear to play important roles in wound contraction in rat fibroblasts (
      • Hirano S.
      • Rees R.S.
      • Gilmont R.R.
      ). Overall, it appears that the primordial MAPK pathways can be used in several permutations, as in the case of ERK, via single or the more recent dual specificity kinases, to achieve the ultimate task of proliferation and differentiation via jun and fos phosphorylation and attachment to the DNA promoter sequence to allow the binding and production of mRNA by RNA polymerase (
      • Kolch W.
      ,
      • Dhanasekaran N.
      • Premkumar R.E.
      ).
      The role of PI3K is similarly diverse in the fibroblast. Cell viability regulation by β1 integrin interaction with the ECM in response to mechanical forces in skin fibroblasts appears to be dependent, at least in part, on PI3K-Akt-protein kinase B signaling pathway activation (
      • Tian B.
      • Lessan K.
      • Kahm J.
      • Kleidon J.
      • Henke C.
      ). PI3K has also been found to be responsible for stimulation of collagen synthesis by active fibroblast cell spreading on culture plates and platelet-derived growth factor-BB (
      • Ivarsson M.
      • McWhirter A.
      • Borg T.K.
      • Rubin K.
      ). In terms of aberrant fibroproliferation, it has been reported that the PI3K pathway is activated in inflammatory lung conditions after lung fibroblast exposure to serum components or effector substances such as insulin-related peptides and transforming growth factor-β to up-regulate α1(I) collagen I mRNA (
      • Ricupero D.A.
      • Poliks C.F.
      • Rishikof D.C.
      • Cuttle K.A.
      • Kuang P.P.
      • Goldstein R.H.
      ).
      In keloids, fibroproliferation was recently shown to be regulated in part by the promitogenic MEK-ERK pathway, signaled by receptor tyrosine kinase phosphorylation by IGF-1. The PI3K-Akt signaling pathway was also found to be activated in synchrony (
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ). In the case of IGF-1, activation of PI3K results in the formation of phosphatidylinositol 3-phosphate, which can serve as a signal for cell growth via its anti-apoptotic influence (
      • Clemmons D.R.
      ), complementing the promitogenic MEK-ERK pathway. This combined effector cascade appears to be quite significant in tumor biology (
      • Gooch J.L.
      • Van Den Berg C.L.
      • Yee D.
      ,
      • Chen H.
      • Yan G.C.
      • Gishizky M.L.
      ).
      Our results confirm our earlier findings (
      • Lim I.J.
      • Phan T.-T.
      • Bay B.H.
      • Qi R.
      • Huynh H.-T.
      • Tan W.T.
      • Lee S.-T.
      • Longaker M.T.
      ,
      • Phan T.-T.
      • Lim I.J.
      • Bay B.H.
      • Qi R.
      • Longaker M.T.
      • Lee S.-T.
      • Huynh H.-T.
      ,
      • Lim I.J.
      • Phan T.-T.
      • Song C.
      • Tan W.T.
      • Longaker M.T.
      ) that KKs do not produce collagen in isolation, that KFs constitutively produce collagens I–III, and that this KF collagen production is increased in co-culture with KKs. The interesting finding of abrogation of collagen I–III production by blocking either MEK1/2 or PI3K with its inhibitor points to a yet unexplored element: the potential of cross-talk between these two pathways. Indeed, this interaction between the MEK-ERK and PI3K pathways appears to affect laminin β2 production to a similar degree, with a significant decrease in laminin β2 protein production upon introduction of the PI3K inhibitor LY294002 and complete abrogation upon the addition of the MEK1/2 inhibitor U0126. Further investigation into the role of the cellular stress- and inflammation-induced JNK and p38 kinase cascades by addition of their inhibitors SP600125 and SB203580, respectively, in a similar co-culture system showed that these pathways do not appear to be involved in collagen and ECM component production in this co-culture model.
      Not all elements of the ECM are affected in the same way, however. In the case of fibronectin, the in-gel migration speed is changed, and we postulate that this might be attributable to loss of glycosylation or glycophosphorylation of the side chains of the molecule. The implication here is that whereas absolute production may not be related to either the MEK-ERK or PI3K pathway, both pathways may play an important role in the post-translational modifications to fibronectin, ultimately affecting its molecular mass. Supporting this last statement are the results from another study in which acetaldehyde-induced fibronectin production in hepatic stellate cells was seen to be inhibited by the addition of calphostin C (a protein kinase C inhibitor), but not by PD98059 (a MEK inhibitor) or wortmannin (a PI3K inhibitor), implying a different intracellular pathway branching downstream of protein kinase C (
      • Svegliati-Baroni G.
      • Ridolfi F.
      • Di Sario A.
      • Saccomanno S.
      • Bendia E.
      • Benedetti A.
      • Greenwel P.
      ).
      Overall, it can be seen that the discrete MEK-ERK and PI3K pathways appear to play a role in the generation of some (but not all) collagen-ECM components in keloids. To our knowledge, this is the first report establishing a link between MEK-ERK and PI3K activation and collagen-ECM production in this fibroproliferative lesion. In addition, it appears that both pathways need to be synchronously activated for collagen I–III and full laminin β2 production. The stress-induced JNK and p38 kinase cascades additionally do not appear to be involved in this fibroproliferative process. A clearer understanding of the intracellular pathways to fibroproliferative scarring will shed more light on this very basic cellular synthetic process and perhaps suggest a new direction for preventing excess collagen-ECM production and deposition in keloids. The identities of the candidate factors triggering keloid fibroblast MEK-ERK and PI3K pathway activation are currently being actively pursued.

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