Cell Communication & Adhesion

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Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro

Julie M. D. Paye a; R. Michael Akers b; William R. Huckle c; Kimberly Forsten-Williams a a Departments of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia b Departments of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia c Departments of Biomedical Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia Online Publication Date: 01 March 2007 To cite this Article: Paye, Julie M. D., Akers, R. Michael, Huckle, William R. and Forsten-Williams, Kimberly (2007) 'Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro', Cell Communication & Adhesion, 14:2, 85 - 98 To link to this article: DOI: 10.1080/15419060701463116 URL: http://dx.doi.org/10.1080/15419060701463116

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Cell Communication and Adhesion, 14: 85–98, 2007 C Informa Healthcare Copyright ISSN: 1541-9061 print / 1543-5180 online DOI: 10.1080/15419060701463116

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro JULIE M. D. PAYE,1 R. MICHAEL AKERS,2 WILLIAM R. HUCKLE,3 and KIMBERLY FORSTEN-WILLIAMS1 Departments of 1 Chemical Engineering; 2 Dairy Science; and 3 Biomedical Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor–I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells–large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I–secreting cells nor addition of exogenous IGF-I or IGF-binding protein–3 reversed the phenol red transport properties. IGF-I–secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels. Keywords autocrine, E-cadherin, epithelial, insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGFBP-2, IGFBP-3, MAC-T, mammary alveolar cells–large T antigen, mammary epithelial cells

velopment (Vleminckx and KemLer 1999), wound healing (Suzuki et al. 2003), and tumor cell metastasis (Beavon 2000). Altered expression and function of junction proteins has been linked to the ability of epithelial cells to migrate (Andre et al. 1999; Li et al. 2001) and metastasize (Bremnes et al. 2002; Kleeff et al. 2001), and increased paracellular permeability has been associated with the formation of tumors (Soler et al. 1999).

INTRODUCTION Epithelial cells are essential for maintaining functional barriers that restrict the transport of proteins and other substances to underlying tissues. This restricted transport is regulated via a series of cell–cell junctions that connect neighboring cells (Nguyen and Neville 1998). These cell–cell connections are altered during various states, such as de-

Received 29 November 2006; accepted 1 May 2007. We appreciate technical assistance from Pat Boyle, Laura J. Delo, and J. Amanda Toepfer. We are grateful for financial support from the National Science Foundation (NSF Career Grant 9875626), Virginia’s Commonwealth Health Research Board, and E.I. DuPont and Company (Young Faculty Award). Address correspondence to Kimberly Forsten-Williams, Department of Chemical Engineering, Virginia Polytechnic Institute and State University, 133 Randolph Hall, Blacksburg, VA 24061. Phone: (540) 231-6631. Fax: (540) 231-5022. E-mail: [email protected] 85


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Expression of proteins that form cell–cell junctions are altered following activation of signaling cascades (Braga 2002; Conacci-Sorrell et al. 2002), such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), including those activated by growth factor receptors (Jones and Kazlauskas 2001). Insulin-like growth factor (IGF-I) is a member of the insulin superfamily and binds with high affinity to its specific cell-surface receptor (IGF-IR) as well as both surface-associated and solution-based binding proteins, known as IGF-binding proteins (IGFBPs). Similarly to other growth factor receptors, IGF-IR is a transmembrane tyrosine kinase that autophosphorylates after ligand binding and can, in many cells, activate signaling cascades, such as PI3K and MAPK (Adams et al. 2000; Ward et al. 2001). Most cancers are of epithelial origin and many of these overexpress IGF-I, insulin-like growth factorII (IGF-II) or IGF-IR (Pollak 2000; Surmacz 2000; Toropainen et al. 1995). The IGF-I system and its receptors are recognized as targets for cancer therapy (reviewed in Sachdev and Yee 2007). For example, expression of a dominant-negative IGF-IR has been shown to reduce metastasis of cells in vivo (Reinmuth et al. 2002; Sachdev et al. 2004). Changes in junction protein expression have been connected to increased tumor cell invasion and metastasis (as reviewed by Mauro et al. (2003)). Activation of IGF-IR has been associated with altered expression of cell– cell junction proteins, such as decreased membrane expression of E-cadherin in human colonic adenocarcinoma cells, as reported by Andre et al. (1999). Moreover, treatment with IGF-I caused sequestration of β-catenin away from E-cadherin in both colorectal and melanoma cancer cells (Playford et al. 2000) following Akt signaling. It is clear that activation of IGF-IR can disrupt cellular junctions and promote migration and metastasis; however, little is known about how autocrine production of IGF-I affects the cell–cell junctions. Our research focused on the relationship between production of IGF-I and increased paracellular permeability. Cell layer permeability was determined by measuring phenol red transport across cell layers of

the mammary epithelial cell line MAC-T (mammary alveolar cell–large T antigen) and two transfected MAC-T cell lines which constitutively secrete IGF-I. Phenol red is a common pH indicator found in most media which is not actively transported across cell layers but which has been used effectively to measure tight junction formation and epithelial layer continuity and shown to be in agreement with tritiated mannitol flux and electrical measurements (Wills 1996; Lewis 2002). The MAC-T cell line formed essentially a complete barrier to the transport of phenol red after six days in culture while both IGF-I secreting cell lines provided essentially no barrier. Neither co-culture studies between MAC-T and the IGF-I secreting cells nor addition of exogenous IGF-I altered parental cell phenol red exclusion properties. Protein levels of both the tight junction protein occludin and the adherens junction protein E-cadherin were reduced in both IGF-I secreting cell lines, as compared with parental cells, suggesting a cellular basis for the reduced barrier properties. Our data suggest that IGF-I, through localized capture of autocrine ligand, accounts for these changes in barrier properties.

METHODS Epithelial Cell Lines The normal bovine mammary epithelial cell line MAC-T (mammary alveolar cells–large T antigen) (Huynh et al. 1991); two IGF-I secreting cell lines (SV40-IGF-I and TK-IGF-I) developed from the MAC-T cell line, transfected to secrete IGF-I under the control of the SV40 and TK promoters respectively (Romagnolo et al. 1994); and plasmid control cell lines, transfected with a plasmid containing the SV40 promoter without IGF-I cDNA; were used in these studies. All cells were grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 50 units/mL of penicillin/streptomycin (Mediatech, Herndon, VA). The plasmid control cell lines were also grown in the


AUTOCRINE IGF-I AND PARACELLULAR TRANSPORT

presence of 0.1 mg/mL hygromycin B (Calbiochem, San Diego, CA). Cell Culture To compare the effects of autocrine and/or exogenous IGF-I (Peprotech, Rocky Hill, NJ) or IGFbinding protein–3 (IGFBP-3; Upstate Biotechnology, Charlottesville, VA) on cell connectivity, cells were cultured in several experimental configurations, either on the upper or lower surface of tissue culture inserts. Additionally, co-culture studies were conducted using one cell type cultured on tissue culture inserts and another cell type cultured in the tissue culture well. In all configurations, cells were R plated at 5 × 104 cells/cm2 on either Transwell inserts (Corning Costar, Cambridge, MA) (12 mm, 0.4 μm pore size) or in twelve-well plates (Corning Costar) and cultured for 8 days, with media replenished every 2–3 days. Note that these inserts have a 0.5 mL inner “apical” container and a 1.5 mL outer “basolateral” container which are separated by the permeable membrane on which the cells are cultured. For exogenous IGF-I or IGFBP-3 studies, proteins were added to both chambers throughout the culture period. Radioimmunoassay for IGF-I Briefly, MAC-T, SV40-IGF-I, and TK-IGF-I cells (2 × 105 ) were seeded and grown in DMEM with 10% FBS for 4 days to reach ∼80% confluence. Thereafter the media was removed and replaced with DMEM without serum (5 mL/well). Medium was harvested after 48 hr (n = 6) and processed essentially as has been previously described elsewhere to determine the concentration of IGF-I (Berry et al. 2001). Phenol Red Transport The redistribution of phenol red across the cell monolayer was evaluated based on a protocol de-

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scribed elsewhere (Wills 1996). Briefly, media was removed from the apical and basolateral chambers and inserts were washed with Dulbecco’s phosphate buffered saline (DPBS). The medium in the apical chamber was replenished with DMEM+ (DMEM with phenol red, 50 units/mL penicillin/streptomycin, and 10% FBS) and the medium in the basolateral chamber was replaced with DMEM– (phenol red–free DMEM, 50 units/mL penicillin/streptomycin, and 10% FBS). Cultures were incubated at 37◦ C for 12 hr. Following incubation, 1 mL samples were taken from the basolateral chamber and analyzed in a spectrophotometer at 479 nm. In the referenced extracellular matrix transport studies, cells were removed from insert as has been described elsewhere (Nugent and Edelman 1992). Total Protein Quantification Total protein quantification based on protein binding to colloidal gold was conducted using Quantigold (Diversified Biotech, Boston, MA) as described by the manufacturer. Briefly, membranes were cut from the insert using a hypodermic needle and incubated in trypsin and EDTA (Mediatech) overnight. Following incubation, buffer (0.1 M Na2 HPO4 , 4 M NaCl, 0.004 M Na2 EDTA–2H2 O; pH 7.4) was added and samples were sonicated at setting 5 for ten seconds using a Model 60 Sonic Dismembrator (Fisher Scientific). Samples were combined with Quantigold and analyzed in a spectrophotometer at 595 nm. Western Ligand Blot Ligand blot analysis was conducted as has been described elsewhere (Hossenlopp et al. 1986). R Briefly, cells were grown on Transwell inserts for eight days with media changed every 2–3 days; conditioned media (CM) was collected on day eight. R CM samples were concentrated using a SpeedVac Model SC110 (Savant, Holbrook, NY) and reconstituted in water. Samples were then diluted with 2x loading buffer (0.125 M Tris, 4% SDS, 20%


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glycerol, 1% bromophenol blue), loaded into a 12.5% acrylamide gel, and separated at constant current. Samples were then transferred to nitrocellulose membranes and incubated overnight with 125 I-IGFI. Membranes were exposed to film for 3–7 days at −70◦ C.

IGF-I Binding Studies R

Cells were plated on Transwell inserts (5 × 104 cells/cm2 ) and cultured for either four or eight days, with media exchanged every 2–3 days. Cells were washed with DPBS, binding buffer (0.05% gelatin, 120 mM NaCl, 5 mM KCl, 1.2-mM MgSO4 , 15 mM sodium acetate, 25 mM Hepes, 10 mM dextrose; pH 7.4) was added to the apical chamber and cells were incubated at 4◦ C for 20 minutes. Following incubation, 125 I-IGF-I (2 ng/mL) and Y60L-IGFI (10 μg/mL) were added and cells were then incubated at 4◦ C for 12 hrs. On ice, binding buffer was aspirated, cells were washed twice with ice-cold DPBS, and lysed with 0.3-N NaOH. Samples were collected and analyzed in a COBRA II Auto-Gamma counter (Perkin–Elmer Life Sciences, Downers Grove, IL).

Western Immunoblot R

Cells were grown on Transwell inserts for eight days with media changed every 2–3 days, collected in Laemmli buffer, and probed with antibodies using Western blot analysis. Samples were sonicated, boiled, and loaded (20 μg total protein per lane for phospho-Akt and total Akt, and 50 μg for all others) into 5% (for E-cadherin), 7% (for occludin, phospho-Akt, and total Akt) or 10% (for IGF-IR and phospho-IGF-IR) Tris–glycine gels. Samples were separated at constant voltage and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were then probed with either rabbit anti– IGF-IRβ (1:10,000) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit (1:15,000) (Zymed, San Francisco, CA); rabbit anti–phospho-IGF-IRβ

(1:1000) (Cell Signaling Technology, Beverly, MA) and anti-rabbit (1:15,000) (Zymed); mouse anti–Ecadherin (1:5000) (BD Biosciences, San Jose CA) and anti-mouse (1:15,000) (Zymed); rabbit antioccludin (1:16,000) (Zymed) and goat anti-rabbit (1:20,000) (Zymed); or rabbit anti–phospho-Akt (1:1000) (Cell Signaling Technology) and goat antirabbit (1:15,000) (Zymed) antibodies. The phosphoAkt blots were stripped (2% SDS, 62.5 mM TrisHCl, 100-mM β-mercaptoethanol; pH 6.7) and reprobed for total Akt using rabbit anti-Akt (1:5000) (Cell Signaling Technology) and goat anti-rabbit (1:15,000) (Zymed) antibodies to ensure that comparable Akt protein levels were found in all lanes. A similar stripping process was done for the phosphoIGF-IR blots. Blots were developed using Pierce Super Signal West Pico Kit (Rockford, IL). Statistical Analysis All experiments were performed a minimum of three times, and the data presented here are representative. Data were evaluated using the GLM procedure of SAS (Version 9.1; SAS Institute, Inc., Cary, NC). For transport data, the main effects of cell type (SV40-IGF-I, MACT, TK-IGF-I, etc.), day (2, 4, 6, and 8), and their interaction, were tested. Results are given as least-square means and standard errors of the mean. Differences between means were compared using the “pdiff ” option of SAS with Tukey’s procedure to declare significance. Significance was defined to be p < 0.05. Binding data were evaluated in a similar manner. Blots are representative of at least three blots run from material obtained from three independent experiments. RESULTS IGF-I–Producing Cells do Not Impede Phenol Red Transport Epithelial cells both in vivo and in vitro are known to form junctions that prevent the unregulated



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Figure 1. IGF-I secreting cells do not inhibit phenol red transport. Phenol red transport across MAC-T (•), SV40-IGF-I (), and TK-IGF-I () cells plated on tissue culture inserts at an initial density of 5 × 104 cells/cm2 was measured, as described in Methods. Measurements (mean ± SEM; n = 4) are normalized to phenol red transport values across control cell free inserts cultured alongside cell inserts. Results are representative of at least three independent experiments.

movement of proteins across the cell barrier (Nguyen and Neville 1998). We investigated transport barrier of MACT cells, a cell line derived from primary bovine mammary epithelial cells (Huynh et al. 1991) using phenol red transport as previously described (Peixoto and Collares Buzato 2005; Forsythe et al. 2002; Simon 2002). MAC-I cells formed a nearly complete barrier to phenol red transport when cultured on tissue culture inserts after day 6 in culture (Figure 1). This barrier function was due to the presence of the MAC-T cells as treatment of the inserts to remove cells but leave extracellular matrix (Nugent and Edelman 1992) resulted in absorbance values which mirrored those of control inserts (inserts maintained in complete media but not seeded with cells) (data not shown). Since IGF-IR is believed to play a role in cell–cell junction formation (Mauro et al. 2001), our hypothesis was that autocrine production of IGF-I, which can affect IGF-IR levels, would alter cell barrier properties. To evaluate this, we tested SV40-IGF-I and TK-IGF-I cells, two MAC-T–derived cell lines stably transfected to secrete IGF-I under the control of two different constitutive promoters (Romagnolo et al. 1994). Expression of IGF-I for the two cell lines was confirmed by radioimmunoassay of conditioned media with aver-

age values of 48.6 ± 2.4 and 9.5 ± 1.0 ng/mL after a 48-hr incubation, for SV40-IGF-I and TK-IGFI cells, respectively. There was no detectible IGF-I ( 0.05 for all days) even though they have vastly different barrier properties (Figure 1). (Note direct overlap of values for MAC-T and TK-IGF-I protein levels on day 8.) Furthermore, SV40-IGF-I and TK-IGF-I cells had identical barrier properties for phenol red at all time points, but significantly different protein levels in cell extracts by day 8 ( p < 0.05). Similar increased densities for SV40-IGF-I were confirmed by DNA analysis (data not shown).

Exogenous IGF-I or IGFBP-3 Does Not Impact MAC-T Barrier Properties Since secretion of IGF-I is the primary difference between the autocrine and parental cells, studies were conducted to determine if addition of exogenous IGF-I could disrupt the ability of MAC-T cells to form a transport barrier. Addition of IGF-I, at levels comparable or greater to those secreted by the autocrine cells, did not alter the ability of MAC-T cells to form a restrictive barrier (Figure 3). However, we also noted differences in the IGF-binding protein profiles from the MAC-T and SV40-IGFI cells cultured on inserts (Figure 4A) similar to those seen on plastic for TK-IGF-I and SV40-IGF-I (Romagnolo et al. 1994). IGFBP-3 was not evident in either the apical or basolateral conditioned media from MAC-T cells; however, it was abundant in the SV40-IGF-I samples (∼250% (apical) and ∼30% (basolateral) more than complete media). We note also the asymmetric distribution of IGFBP-3 between the apical and basolateral chambers, consistent with the existence of a diffusional barrier to larger molecules across the monolayer of IGF-I– secreting cells despite the lack of transport barrier for phenol red. Given that IGFBP-3 has been shown to



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Figure 3. Exogenous IGF-I does not affect phenol red transport across MAC-T cells. Phenol red transport across MAC-T cultured in complete DMEM supplemented every media change with 0 (•), 50 (), or 100 ng/mL () IGF-I plated on tissue culture inserts at an initial density of 5 × 104 cells/cm2 . Measurements (mean ± SEM; n = 4) are representative of three independent experiments.

Figure 4. Exogenous IGFBP-3 does not affect phenol red transport across MAC-T cells. (A) Western ligand blot of MAC-T– and SV40-IGFI–conditioned media and control (complete medium not exposed to any cells). Conditioned media were collected from the apical (0.5 mL) and basolateral (1.5 mL) compartments of the Transwell and treated as described in Methods. Bands corresponding to IGFBP-3 and IGFBP-2 are shown. Blots are representative of three independent experiments. (B) Phenol red transport across MAC-T incubated with 0 (•), 10 (), 100 (), and 1000 () ng/mL IGFBP-3. Measurements (mean ± SEM; n = 4) are representative of three independent experiments. Note that results are shown for each treatment on days 2, 4, 6, and 8 but are difficult to visualize due to overlap of mean values.


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exhibit IGF-I–independent properties, MAC-T cells were plated and incubated in complete media supplemented with recombinant IGFBP-3. Inclusion of exogenous IGFBP-3 did not impair the ability of MACT cells to form a restrictive barrier (Figure 4B). We also noted that addition of exogenous recombinant IGFBP-3 to both TK-IGF-I and SV40-IGF-I cell cultures did not alter/increase the barrier properties of these cells (data not shown).

Co-Culture Does Not Dramatically Alter Barrier Properties of MAC-T or SV40-IGF-I Cells IGF-I can induce secretion of a number of other effectors, such as vascular endothelial growth factor (VEGF; Burroughs et al. 2003) and estradiol (Kolodziejczyk et al. 2003). For example, Cohick and Turner (1998) found IGFBP-6 as well as IGFBP2 in conditioned media from MAC-T cultured on tissue culture plastic; and Romagnolo et al. (1994) showed that MAC-T cells cultured on plastic were stimulated by conditioned media from both TK-IGFI and SV40-IGF-I cells, indicating cell responsive-

ness to released factors. We observed that SV40IGF-I cells secreted ∼15% more apical and ∼40% more basolateral IGFBP-2 than MAC-T cells, in addition to the difference in IGFBP-3 secretion (Figure 4A; note differences in the levels of IGFBP2 and IGFBP-3 between the apical and basolateral chambers from SV40-IGF-I–conditioned media, indicating that the lack of phenol red barrier was not due to gross changes in the monolayer continuity). Co-culture systems were utilized to address whether these other factors, or a specific combination of factors, might be responsible for the loss of phenol red exclusion exhibited by the IGF-I-secreting cells. Inclusion of SV40-IGF-I cells within the culture (well surface) only marginally affected phenol red exclusion by MAC-T cells, exhibiting the same general trend for phenol red exclusion with time found as in single culture (Figure 5 and Table 1). Similarly, inclusion of MAC-T cells did not greatly alter the poor barrier properties of SV40-IGF-I ( p > 0.05 for days 2, 4, and 6; p = 0.05 for day 8), suggesting that a “barrier-promoting” factor was not being secreted by the parental cell line. Furthermore, only a small non-significant difference ( p > 0.05 for days 6 and

Figure 5. Co-culturing has non-significant effect on phenol red transport across SV40-IGF-I and MAC-T cells. Normalized phenol red transport across MAC-T (•), SV40-IGF-I (), MAC-T (grown on insert) co-cultured with SV40-IGF-I cells (grown on well surface) (◦), and SV40-IGF-I (grown on insert) co-cultured with MAC-T (grown on well surface) (), measured as described in Methods. Measurements (mean ± SEM; n = 4) are normalized to phenol red transport values across control cell free inserts cultured alongside cell inserts. Results are representative of at least three independent experiments.


AUTOCRINE IGF-I AND PARACELLULAR TRANSPORT TABLE 1 Phenol red transport across MAC-T in single culture and in co-culture with SV40-IGF-I cells. Measurements (mean ± SEM; n = 4) are representative of three independent experiments and are normalized to percentage of phenol red absorbance of media from inserts without cells. Apical surface Days of growth 2 4 6 8

Basolateral surface

Single culture

Co-culture

Single culture

Co-culture

68% ± 3% 20% ± 3% 7% ± 3% 4% ± 3%

74% ± 3% 23% ± 3% 26% ± 3%∗ 13% ± 3%

93% ± 3%∗ 40% ± 3%∗ 15% ± 3% 16% ± 3%

98% ± 3%∗ 39% ± 3%∗ 24% ± 3%∗ 29% ± 3%∗

∗ Significantly different ( p

< 0.05) from single culture apical surface

on corresponding day.

8) in phenol red exclusion between MAC-T cells cultured on the lower (basolateral) or upper (apical) surface of the insert was found (Table 1) suggesting the insert surface properties were not critical for the response. A small (when compared to the difference between MAC-T and SV40), but significant ( p < 0.05 for all days), difference in barrier properties was found between MAC-T single apical culture and co-culture samples with MAC-T cultured on the basolateral surface. We note, however, that differences in barrier properties were minor ( p ≥ 0.05) between MAC-T cultured on the basolateral surface in single and co-culture samples, suggesting negligible polarity in response to factors secreted by cells on the tissue culture well.

Decreased IGF-IR Binding for Parental Cells at Day 8 but Unchanged Levels for Igf-I Secreting Cells We next asked whether differences in IGF-IR levels were evident between MAC-T cells and IGF-I– secreting cells in our culture system and if receptors on the MAC-T cells were sensitive to IGF-I. In previous work, when cultured on tissue culture plastic, the SV40-IGF-I cells had reduced IGF-IR binding when compared to the MAC-T cells while TK-IGF-I cells had similar levels (Romagnolo et al.

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1994). Receptor-binding studies were conducted in the presence of excess Y60L-IGF-I, an analogue with normal affinity to IGFBPs but minimal affinity for IGF-IR (Bayne et al. 1990), to block binding of 125 I-IGF-I to IGFBPs (Figure 6A). MAC-T cells bound significantly ( p < 0.05) more 125 I-IGF-I per cell than either of the IGF-I secreting cell lines at day 4; however, there was significantly less IGF-IR binding per cell for the parental cells than TK-IGF-I on day 8 ( p < 0.05). This change was not due to an increase in IGF-IR capacity for the IGF-I–secreting cell lines but to a decrease (per mg of protein) for the parental cells. TK-IGF-I cells exhibited significantly more IGF-IR binding than did SV40-IGF-I cells ( p < 0.05), and neither cell line showed a significant difference between four and eight days ( p > 0.05). Verification that binding correlated with IGFIR protein was obtained via Western analysis of total cell lysate, where less intense bands were found for MAC-T cell lysate when probed for total IGF-IR (Figure 6B). Despite the reduced levels found, IGFIRs on the parental as well as on the autocrine cells were responsive to IGF-I as evident by phosphorylation of IGF-IR (Figure 6B) and Akt (Figure 6C) upon exposure to exogenous IGF-I for 10 min (note that the reduced levels of IGF-I on the MAC-T necessitated the showing of different exposures of blots for the phosphorylated IGF-IR). This responsiveness of the receptor to IGF-I indicated that the lack of change in permeability of the MAC-T cell layer on exposure to exogenous IGF-I (Figure 3) was not due to a defect or inability of the receptor to activate. Certainly, however, the difference in IGF-IR levels could result in differences in responsiveness to IGF-I, potentially illustrated by the differences in phosphorylated Akt. We note that phosphorylation of ERK following IGF-I exposure was not evident in any of the cell lines, in agreement with previous work by Grill et al. (2002) with MAC-T cells (data not shown). Furthermore, some basal phosphorylation of Akt was seen with the SV40-IGF-I cells not evident with either the TK-IGF-I cells or MAC-T cells, suggesting it was not required or responsible for the change in phenol red permeability of the IGF-I cell line.


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Figure 6. Comparison of IGF-IR levels in IGF-I–secreting and MAC-T cells with stimulation with IGF-I. (A) Binding of 125 I-IGF-I to MAC-T (), SV40-IGF-I (), and TK-IGF-I (gray) cells at 4◦ C after four or eight days in culture, as described in Methods. Measurements (mean ± SEM; n = 2) are representative of three independent experiments. (B) Western immunoblot of MAC-T, SV40-IGF-I, and TK-IGF-I total cell lysates, probed for IGF-IR β and phospho-IGF-IR β on day 8 after being treated for 10 min with IGF-I (0 or 100 ng/mL). (C) Western immunoblot of MAC-T, SV40-IGF-I, and TK-IGF-I cell lysates probed for phospho-Akt on day 8 after being exposed to IGF-I (0 or 100 ng/mL) for 10 min. All blots (B and C) are representative of blot results from three independent cell experiments and were loaded with equivalent amounts of protein.

IGF-I Producing Cells Express Reduced Levels of Adherens and Tight Junction Proteins Since cell–cell junctions regulate paracellular transport barriers, Western immunoblots were conducted to identify key junction proteins associated with the differences in barrier properties between parental and IGF-I–secreting cells. A reduction of ∼50% in the level of the tight junction protein occludin, when compared to the MAC-T cells, was found for the IGF-I–secreting cell lines (Figure 7A) with both the SV40-IGF-I and TK-IGF-I lines having similar levels of occludin on an equal protein basis. Furthermore, expression of the adherens junction protein E-cadherin was also reduced for the IGFI–secreting cell lines when compared to the parental

Figure 7. IGF-I secreting cells have decreased levels of occludin and E-cadherin. Western immunoblots of MAC-T, SV40-IGF-I, and TK-IGF-I cell lysates from cells plated on tissue culture inserts at 5 × 104 cells/cm2 , lysed after 8 days of culture, and probed for (A) occludin and (B) E-cadherin, as described in Methods. Note that lanes were loaded with equal micrograms of total protein. Arrows indicate the expected molecular weights of occludin (65 kDa) and E-cadherin (120 kDa). Blots are representative of blot results from three independent cell experiments.



cell line (Figure 7B). The reduction was particularly dramatic for the SV40-IGF-I cells, with an approximately 90% reduction in E-cadherin on an equal micrograms of total protein basis and an approximately 20% reduction for TK-IGF-I cells. We note that a lower-molecular-weight band was found for E-cadherin in cell lysates from all three cell lines which did not appear to be differentially regulated between the IGF-I secreting lines and the MAC-T line. A similarly sized smaller band for E-cadherin has been seen from cell lysate of other epithelial cells (Husmark et al. 2002; Burke and Hong 2006) and has been suggested to be due to post-transcriptional modifications.

DISCUSSION Growth factors and their receptors, including IGFI and IGF-IR, are capable of altering cell barrier formation (Hellawell et al. 2002; Pennisi et al. 2002; Toropainen et al. 1995) although the effect appears to be cell type–specific. For example, McRoberts et al. (1992) report increased permeability in the T84 human colonic epithelial cell line after two days of basolateral exposure to IGF-I. Ericson and Nilsson (1996) did not detect any significant increase in permeability of pig thyrocytes until after 16 days of treatment with IGF-I, and the increase was only a partial loss of barrier properties, not the nearly complete loss reported by McRoberts and Riley (1992). In this report, we find no effect of exogenous IGF-I on the paracellular permeability of the bovine mammary epithelial cell line, MAC-T, to phenol red despite a dramatic difference in permeability evident in two transfected MAC-T cell lines which secrete IGF-I. Our data suggest that the difference is due to an effect of locally produced IGF-I. Various growth factors, such as fibroblast growth factor (FGF) (Reuss et al. 2003), transforming growth factor (Lui et al. 2003), and hepatocyte growth factor (Jiang et al. 1999) have been reported to decrease levels of occludin, resulting in increased paracellular permeability. Recent work by Spoerri et al. (2006), using human retinal endothelial cells,

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showed that high glucose culture increased the level of IGF-I in conditioned media, and also resulted in decreased occludin levels. Transfection of these cells with hammerhead ribozymes targeted at IGF-IR reversed the occludin level reduction, suggesting that autocrine IGF-I activation of IGF-IR was responsible for the reduced occludin levels. In agreement, we have found that our IGF-I secreting cells expressed lower levels of occludin and demonstrated increased permeability to phenol red. Recent studies have also suggested a relationship between increased paracellular permeability and expression of cadherins (Friedl et al. 2002; Nwariaku et al. 2002; Tan et al. 2000; Venkiteswaran et al. 2002). Although associated with adherens junctions, E-cadherin can colocalize at tight junctions (West et al. 2002), which have traditionally been thought to regulate paracellular transport (Stevenson and Keon 1998). Zabner et al. (2003) reported that treatment of various types of epithelial cells with histamine resulted in increased permeability that was attributed to a reduction in E-cadherin–mediated junctions. Furthermore, blocking peptides to the EC1 domain of E-cadherin produced a three-fold increase in the permeability of Madin-Darby canine kidney epithelial cell layers (Sinaga et al. 2002). In this investigation, we showed that two IGF-I– secreting cell lines developed with independent plasmids had less E-cadherin protein than the non–IGFI-secreting parental cell line, which was coincident with increased transport of the paracellular permeability marker phenol red and decreased IGF-IR levels. These results suggest that the reduced levels of E-cadherin or occludin are responsible for the increased paracellular permeability; however, future knockdown studies in MAC-T and fluorescent imaging of junctions would be beneficial. Growth factor action can occur through several different mechanisms. In autocrine signaling, growth factors are secreted by cells and released to the extracellular environment, where they bind to and activate growth factor receptors on the cell surface (Sporn and Roberts 1992). However, with intracrine signaling, stimulation occurs before the growth factor can be released to the extracellular


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environment via interaction with intracellular receptors (Re 2002). In recent years, there has been a growing interest in intracrine signaling, particularly with regard to the endothelial growth factor (EGF) (Wiley et al. 1998), FGF (Bilak et al. 2003; Stachowiak et al. 1997), angiotensin II (Baker et al. 2004) and IGF (Dubois et al. 1993; Lin et al. 1997; Van der Ven et al. 1997; Baumrucker 2005) families. In our studies, addition of exogenous growth factors or other effectors, such as IGFBP-3, failed to alter the barrier properties of parental cells; likewise, addition of IGFBP-3 in order to bind endogenously secreted IGF-I did not impact the IGF-I–secreting cell line barrier properties. This suggests that either localized autocrine stimulation or intracrine actions could be the mechanism of action. Work by Cohick in collaboration with others (Grill and Cohick 2000; Grill et al. 2002), indicated that MACT cells transfected to secrete IGFBP-3 showed altered response to exogenous IGF-I in comparison with parental cells exposed to IGF-I and exogenous IGFBP-3, supporting the idea of localized autocrine or intracrine signaling being possible within the IGF axis. Moreover, previous studies have shown that inhibition of autocrine ligand binding is difficult and may require unattainable concentrations of soluble inhibitors (i.e., IGFBP-3 or other IGF-I–binding proteins) to eliminate receptor binding and activation (Forsten and Lauffenburger 1992a; Forsten and Lauffenburger 1992b; DeWitt et al. 2001). We cannot at this time determine whether the effects we have noted are due to secreted IGF-I acting proximally to the cell surface in a way that exogenous IGF-I in our system cannot, or if the interaction occurs intracellularly through an intracrine mechanism. Striking differences in both the IGF-IR levels, and the change over time of those levels, were found between the parental and IGF-I–secreting cells, but how these differences might make manifest a change in paracellular transport is unclear. For example, some studies indicate metastasis correlates with reducing IGF-IR levels (Nakamura et al. 2004; Pennisi et al. 2002; Schnarr et al. 2000), while others indicate inhibition of metastasis by reducing IGF-IR levels (Dunn et al. 1998; Sachdev et al. 2004). It is also conceivable

that IGF-I–induced IGFBP-3 may be responsible, as this protein has been suggested to have intracrine activity (Baumrucker 2005). Additional studies, using siRNA to alter, variously, IGF-I, IGFBP-3, and IGFIR levels, are needed to discern specific mechanisms of action. In conclusion, the non–IGF-I-secreting cell line MAC-T formed a nearly exclusive transport barrier to phenol red, in contrast to the lack of barrier found from two independently developed IGF-I secreting cell lines. Addition of exogenous IGF-I, or co-culturing with the IGF-I–secreting cell lines, did not lead to the same lack of phenol red barrier evident with the TK-IGF-I and SV40-IGF-I cells. All cells expressed IGF-IR and were responsive to IGF-I. However, the IGF-IR levels differed, as did the levels of the cell–cell junction components, E-cadherin, and occludin. Our results suggest that production of IGF-I somehow alters the levels of important junctional proteins which impact the overall connectivity of cells in culture, and provide the foundation for further studies targeted at deciphering the signaling mechanisms involved in this process. REFERENCES Adams TE, Epa VC, Garrett TP, Ward CW (2000). Structure and function of the type 1 insulin-like growth factor receptor. Cell Mol Life Sci 57: 1050–1093. Andre F, Rigot V, Thimonier J, Montixi C, Parat F, Pommier G, Marvaldi J, Luis J (1999). Integrins and E-cadherin cooperate with IGF-I to induce migration of epithelial colonic cells. Int J Cancer 83: 497–505. Baker KM, Chernin MI, Schreiber T, Sanghi S, Haiderzaidi S, Booz GW, Dostal DE, Kumar R (2004). Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy. Regul Pept 120: 5–13. Baumrucker CR (2005). Intracrine signaling in the mammary gland. Livestock Production Science 98: 47–56. Bayne ML, Applebaum J, Chicchi GG, Miller RE, Cascieri MA (1990). The roles of tyrosines 24, 31, and 60 in the high affinity binding of insulin-like growth factor-I to the type 1 insulin-like growth factor receptor. J Biol Chem 265: 15648–15652. Beavon IR (2000). The E-cadherin-catenin complex in tumour metastasis: structure, function and regulation. Eur J Cancer 36: 1607– 1620. Berry SD, TB McFadden, RE Pearson, RM Akers (2001). A local increase in the mammary IGF-I: IGFBP-3 ratio mediates the mammogenic effects of estrogen and growth hormone. Domest Anim Endocrinol 21: 39–53. Bilak MM, Hossain WA, Morest DK (2003). Intracellular fibroblast growth factor produces effects different from those of extracellular


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