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Fatty acids modulate transforming growth factor-ß activity and plasma clearance ¹

THAI-YEN LING, YEN-HUA HUANG^*, MING-CHIH LAI, SHUAN SHIAN HUANG and JUNG SAN HUANG^,,2

Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;
^* Department of Biochemistry, Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan; and
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

²Correspondence: Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, USA. E-mail: huangjs{at}slu.edu or huangss{at}slu.edu

SPECIFIC AIMS

Activated ₂M (₂M*) is known to regulate the activity and plasma clearance of TGF-ß by complexation with TGF-ß, which involves hydrophobic interactions with topologically exposed molecular surfaces. Small hydrophobic compounds may be capable of blocking and/or dissociating TGF-ß–₂M* complexes, thereby modulating TGF-ß activity and plasma clearance. The aim of this study was to determine the effects of fatty acids on complex formation of TGF-ß isoforms and ₂M* on ₂M* inhibition of TGF-ß binding, TGF-ß-induced growth inhibition, and TGF-ß-induced transcriptional activation in mink lung epithelial cells and on plasma clearance of TGF-ß–₂M* complexes in mice.

PRINCIPAL FINDINGS

1. Fatty acids inhibit complex formation of TGF-ß isoforms and ₂M* and are capable of dissociating TGF-ß–₂M* complexes
We screened many small hydrophobic compounds present in plasma and tissue for their ability to inhibit complex formation of ¹²⁵I-TGF-ß isoforms and ₂M*. This involved 5% nondenaturing PAGE and autoradiography, a standard method for determining complex formation of ¹²⁵I-TGF-ß and ₂M*. Among the screened agents, only fatty acids had this property, suggesting they may be important modulators of the interaction between TGF-ß and ₂M* in vivo. The ability of fatty acids to inhibit complex formation of ¹²⁵I-TGF-ß isoforms and ₂M* was found to depend on carbon chain length (C20, C18, C16, C14>C12>C10), degree of unsaturation (polyunsaturated>saturated), and TGF-ß isoforms (TGF-ß₁>TGF-ß₂>TGF-ß₃). The IC₅₀s of saturated fatty acids ranged from 7–9 µM (stearic acid/palmitic acid/myristic acid) to >100 µM (caprylic acid). Unsaturated fatty acids exhibited IC₅₀s of 5–8 µM (arachidonic acid/oleic acid/-linolenic acid/linoleic acid), 15 µM (palmitoleic acid), and 26 µM (linoleic acid). Arachidonic acid, one of the most potent inhibitors, was also capable of dissociating ¹²⁵I-TGF-ß–₂M* complexes but higher concentrations were required. Arachidonic acid appeared to inhibit ¹²⁵I-TGF-ß-₂M* complex formation by binding specifically to ₂M* as determined by gel filtration chromatography.

2. Fatty acids block the inhibitory effect of ₂M* on TGF-ß binding to TGF-ß receptors, TGF-ß-induced growth inhibition, and TGF-ß-induced transcriptional activation in mink lung epithelial cells (Mv1Lu)
We determined the effects of arachidonic acid on ¹²⁵I-TGF-ß₂ binding (in the presence and absence of ₂M*) to Mv1Lu cells. ₂M* is known to inhibit TGF-ß₂ more strongly than TGF-ß₁ binding to TGF-ß receptors in cells. Various concentrations of ¹²⁵I-TGF-ß₂ were preincubated with 200 µg/mL of ₂M* in the presence or absence of 30 µM arachidonic acid for 30 min prior to the performance of binding assays in Mv1Lu cells. As shown in Fig. 1 A, ₂M* strongly inhibited ¹²⁵I-TGF-ß₂ binding to Mv1Lu cells. The residual ¹²⁵I-TGF-ß binding associated with the cells after ₂M* inhibition was mainly due to nonspecific binding of ¹²⁵I-TGF-ß₂. ₂M* at 200 µg/mL completely inhibited specific binding of ¹²⁵I-TGF-ß₂ to those epithelial cells, as previously reported. The inhibition by ₂M* was completely reversed by 30 µM of arachidonic acid. To clarify the biological relevance of this observation, we determined the effect of arachidonic acid on the inhibitory effect of ₂M* on TGF-ß₂-induced growth inhibition and TGF-ß₂-induced transcriptional activation in Mv1Lu cells. ₂M* has been shown to be effective in blocking TGF-ß₂-induced growth inhibition. As shown in Fig. 1B , TGF-ß₂ inhibited [methyl-³H]-thymidine incorporation into DNA of Mv1Lu cells in a dose-dependent manner. In the presence of 200 µg/mL of ₂M*, the dose-response curve of TGF-ß₂ shifted to the right. In the absence of ₂M*, TGF-ß₂ (1 pM) inhibited 25% of [methyl-³H]-thymidine incorporation into DNA of these epithelial cells; this was completely abolished by the presence of ₂M* in the medium. Addition of arachidonic acid at 0.5 and 1 µM reversed the inhibitory effect of ₂M* on TGF-ß₂-induced growth inhibition as measured by [methyl-³H]-thymidine incorporation. One µM of arachidonic acid almost completely reversed the inhibitory effect of 2M* on growth inhibition induced by 1 pM of TGF-ß₂. In the absence of ₂M*, arachidonic acid did not affect growth inhibition induced by TGF-ß₂ under the experimental conditions.

View larger version (23K): [in this window] [in a new window]   Figure 1. Arachidonic acid reversal of the 2M* inhibitory effect on 125I-TGF-ß2 binding to TGF-ß receptors (A), TGF-ß2-induced growth inhibition (B), and TGF-ß2-induced transcriptional activation (C) in Mv1Lu cells. A) 2M* (200 µg/mL) was preincubated with arachidonic acid (AA) (0 or 30 µM) and various concentrations (0, 1.25, 2.5, 5, and 10 pM) of 125I-TGF-ß2 with and without TGF-ß peptantagonist (30 µM) in binding buffer (50 mM HEPES/NaOH, pH 7.4, 128 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.2 mM CaCl2) containing 2 mg/mL of bovine serum albumin. After 30 min at room temperature, the 125I-TGF-ß2 solution was added to cells and 125I-TGF-ß2 binding was determined after 2.5 h at 0°C. The binding of 125I-TGF-ß2 obtained in the presence of 2M* was mainly nonspecific binding of 125I-TGF-ß since it was not further inhibited by the presence of TGF-ß peptantagonist. Data are representative of 4 similar experiments. B) Cells were treated with various concentrations of TGF-ß2 in the presence and absence of 2M* (200 µg/mL) and arachidonic acid (AA) (0.5 and 1.0 µM) in DMEM containing 0.2% fetal calf serum (FCS). After 18 h at 37°C, the [methyl-3H]-thymidine incorporation into cellular DNA of cells was determined. The [methyl-3H]-thymidine incorporation in cells treated without TGF-ß2 and arachidonic acid was taken as 0% inhibition. Data are representative of 4 similar experiments. C) Cells transiently transfected with the p3TP plasmid were treated with various concentrations of TGF-ß2 in the presence and absence of 2M* (200 µg/mL) and arachidonic acid (AA) (12.5 and 25 µM) in DMEM containing 0.2% FCS. After 12 h at 37°C, the luciferase activity of the cell extracts was determined and expressed as arbitrary units (A.U.). Data were obtained from three different experiments; values are mean ± SD (*P<0.05 vs. luciferase activity of cells treated with 2M* and TGF-ß2).

We then determined the effect of fatty acids on the inhibition by ₂M* of the expression of a TGF-ß-responsive promoter construct p3TP-Lux in transfected Mv1Lu cells. The p3TP-Lux contains the PAI-1 promoter and three repeats of a phorbol-12-myristate-13-acetate (TPA) -responsive element. As shown in Fig. 1C , ₂M* (200 µg/mL) inhibited 25–30% of the luciferase activity induced by TGF-ß₂ (50 and 100 pM). This ₂M* inhibition of the TGF-ß-induced luciferase activity was reversed by either 12.5 or 25 µM of arachidonic acid. In the control experiments, arachidonic acid (12.5 and 25 µM) did not influence luciferase activity in the cells treated with or without TGF-ß₂ in the absence of ₂M* (data not shown). With the results described above, this suggests that fatty acids are capable of modulating the biological activities of TGF-ß when ₂M* is present.

3. Fatty acids block ₂M*-mediated plasma clearance of TGF-ß₁ and TGF-ß₂
Since fatty acids are able to block complex formation of TGF-ß and ₂M*, they might be able to affect the plasma clearance of TGF-ß and ₂M* complexes. To test this, ¹²⁵I-TGF-ß₁ or ¹²⁵I-TGF-ß₂ were preincubated with ₂M* in the presence or absence of 10 µM arachidonic acid at room temperature for 30 min, then injected into mice via tail vein according to published procedures. At several intervals (10 s, 1, 2, 3, 5, 10, 15, 20, 30, and 60 min) 25 µL of blood was collected and counted by a -counter. As shown in Fig. 2 A, B, the estimated plasma clearance half-times (t_1/2s) of free ¹²⁵I-TGF-ß₁ (Fig. 2A ) and ¹²⁵I-TGF-ß₂ (Fig. 2B ) were 1.8 ± 0.2 (n=4) and 1.3 ± 0.3 (n=4) min, respectively. The t_1/2s of ¹²⁵I-TGF-ß₁+₂M* and ¹²⁵I-TGF-ß₂+₂M* were 3.8 ± 0.2 (n=4) and 3.7 ± 0.1 (n=4) min, respectively. These t_1/2s are consistent with published values of free ¹²⁵I-TGF-ß_1,2 and ¹²⁵I-TGF-ß_1,2–2M* complexes. In the presence of arachidonic acid, the t_1/2s of ¹²⁵I-TGF-ß₁ + ₂M* and ¹²⁵I-TGF-ß₂+₂M* were decreased to 1.9 ± 0.1 (n=4) and 1.8 ± 0.2 (n=4) min, respectively; these are essentially identical to the t_1/2s of free ¹²⁵I-TGF-ß₁ and ¹²⁵I-TGF-ß₂ (Fig. 2A, B ). In control experiments, arachidonic acid did not affect the plasma clearance of free ¹²⁵I-TGF-ß₁ and ¹²⁵I-TGF-ß₂ (data not shown). There results suggest that arachidonic acid may be capable of affecting the plasma clearance of TGF-ß + ₂M* by blocking complex formation.

View larger version (21K): [in this window] [in a new window]   Figure 2. Plasma clearance of 125I-TGF-ß1 (A) or 125I-TGF-ß2 (B) treated with 2M* in the presence and absence of arachidonic acid 125I-TGF-ß1 (A) or 125I-TGF-ß2 (B) was incubated with 2M* in the presence and absence of arachidonic acid (AA). After 30 min at room temperature, the 125I-TGF-ß1 or 125I-TGF-ß2 solution was injected into the tail veins of mice. Blood samples were collected at the intervals indicated. The radioactivity in the blood sample collected 10 s after i.v. injection of the isotope solution was taken as 100%. Data are representative of 4 similar experiments.

CONCLUSIONS AND SIGNIFICANCE

Low levels of active TGF-ß in plasma and tissues have been implicated in the pathogenesis of atherosclerosis, autoimmune disease, and malignancy (Fig. 3 ). Compounds capable of blocking and/or dissociating TGF-ß–₂M* complexes, thereby affecting the levels of free active TGF-ß in plasma and tissues, have therapeutic potential as systemic or regionally delivered drugs for these diseases. Here we demonstrate that fatty acids are potent inhibitors of complex formation of TGF-ß and ₂M*. The IC₅₀s of most of the fatty acid examined for inhibiting TGF-ß binding to ₂M* are <10 µM. These concentrations can occur at sites of injury (wound) or inflammation. Fatty acids are known to be generated locally at considerably higher concentrations than the mean blood levels. In the interstitial space, where albumin concentration is much lower than within the blood, fatty acids may modulate TGF-ß activity even more significantly than in plasma. Fatty acids (e.g., arachidonic acid) have also been found to block complex formation between ₂M* and nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) in the laboratory (unpublished results). This suggests that exogenous fatty acids (e.g., polyunsaturated fatty acids) can be designed to potentiate TGF-ß and other growth factor/cytokine/hormone activities in order to treat human or animal diseases (Fig. 3) .

View larger version (11K): [in this window] [in a new window]   Figure 3. Fatty acids potentiate TGF-ß activity by influencing complex formation between TGF-ß and 2M*. Low levels of TGF-ß in plasma and tissues are associated with certain human diseases, e.g., atherosclerosis, autoimmune diseases, and malignancy. Exogenous fatty acids, especially polyunsaturated fatty acids, can be designed to potentiate TGF-ß activity by blocking and/or dissociating TGF-ß–2M* complexes. This suggests novel ways of treating these diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1063fje; doi: fj.02-1063fje

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