The effect of mannose-6-phosphate in reducing transforming growth factor proliferation of McCoy fibroblast cells.
Tendon healing is plagued by the complications of rupture and adhesion formation. Both of these tend to occur early in healing and are due to improper regulation of collagen formation. Flexor tendon repairs are plagued by the formation of peripheral adhesions that limit motion. An important aspect of tendon healing is the timing and degree of collagen expression during the repair process. Connective tissue growth factor (CTGF) is a member of a family of regulatory proteins, which along with transforming growth factor beta (TGF-[beta]), plays a central role in Type I collagen and extracellular matrix (ECM) production . Over-expression of CTGF has been noted in multiple fibrotic tissues [2-5]. Increased expression of CTGF is enhanced by placing tissues under hypoxic conditions . CTGF has been shown to up-regulate TGF-[beta], which is the major player in fibrosis through induction of ECM fabrication, and is additionally recognized to play an important role in continued signaling for CTGF . This cooperative role of TGF-[beta] and CTGF in fibrotic pathways presents a viable target for therapy in tissue fibrosis.
Our lab has shown that fructose-1,6-bisphosphate (FBP) is an intermediate in glycolysis which, if provided to cells, could allow them to bypass two ATP-requiring steps for energy production. Huang and colleagues have previously shown that treatment of cells placed under hypoxic conditions with FBP decreases production of CTGF to near control conditions . Mannose-6-phosphate (M6P), a natural sugar, is known to isomerize to fructose-6-phosphate (F6P), an intermediate of glycolysis in the step prior to FBP. Therefore, one might hypothesize that treatment with M6P may decrease production of CTGF by decreasing the need for substrate-level phosphorylation, which would be vital in hypoxic conditions. Alternatively, latent TGF-[beta] is activated through its binding with the M6P/IGF-II receptor . M6P has been shown to interact with the M6P/IGF-II receptor on fibroblasts. Therefore, competitive inhibition of the activation of TGF-[beta] through treatment with M6P is considered another method by which M6P may inhibit fibrosis. It is thought that M6P may have more potential for reducing fibrosis than FBP because its mechanism of action is not dependent upon a hypoxic environment.
Since M6P acts at the same receptor as TGF-[beta]1 on fibroblasts, it is possible that treatment with M6P may inhibit fibrosis. Our goal was to determine if M6P could prevent or reduce TGF-[beta]1-induced fibroblast proliferation. This study is clinically important since no current adjunct therapy is available to prevent scar formation.
Cell Culture: McCoy [McCoyB] (ATCC[R] CRL-i696[TM]) fibroblasts were obtained from American Type Culture Collection (ATCC, Rockville, MD) and supplemented with Dulbecco's Modified Eagle's Medium (DMEM) + 5% fetal bovine serum + 1% antibiotic/antimycotic solution in T-75 culture flasks where they were grown to confluence under normoxic conditions at 37[degrees]C.
Cell Culture for M6P Concentration Studies: Once confluent, the cells were plated on 24-well plates where row A served as a control, row B served as 0.005 [micro]M (Low) M6P (Sigma-Aldrich) treatment, row C served as 0.05 [micro]M (Medium) M6P treatment, and row D served as 0.5 [micro]M (High) M6P treatment. Columns 5 and 6 of each plate contained coverslips for staining.
Cell Culture for TGF-pi Concentration Studies: Once confluent, the cells were plated on 24-well plates where row A served as a control, row B served as 200 ng (Low) TGF-[beta]1 (Sigma-Aldrich) treatment, row C served as 1,000 ng (Medium) TGF-[beta]1 treatment, and row D served as 2,000 ng (High) TGF-[beta]1 treatment. Columns 5 and 6 of each plate contained coverslips for staining.
Cell Culture for Competitive Study: Once confluent, the cells were plated on 24-well plates where row A served as a control, row B served as 0.05 [micro]M (Medium) M6P (Sigma-Aldrich) treatment, row C served as 200 ng (Low) TGF-[beta]1 (Sigma-Aldrich) treatment, and row D served as the M6P/TGF-[beta]1 combination treatment. Columns 5 and 6 of each plate contained coverslips for staining.
Cell Count: At the end of each 24, 48, and 72 hour incubation period, cell counts were performed using standard hemacytometer techniques. Briefly, an aliquot of the cell suspension was diluted in a 1:1 volume of trypan blue prior to adding 10 [micro]l of cell/dye mixture to each chamber of the hemacytometer. The average number of cells per square was multiplied by the dilution factor prior to multiplying it by 10,000 to determine the average number of cells per milliliter.
Glutathione (GSH) Assay: Oxidative damage to the cells was assessed by utilizing an enzyme assay to determine glutathione peroxidase levels and subsequently estimate glutathione levels. Decreases in cellular glutathione concentrations correlate directly with cellular viability. Briefly, 0.05 mL of standard and samples were placed in corresponding wells of a 96-well plate. Then, 0.1 mL of reaction mixture consisting of 5 mL DNTB, 5 mL NADPH, 5.75 mL buffer solution, and 0.01 mL GSH reductase was added to each well. The plate was placed in a microtiter plate reader and the absorbances were measured at a wavelength of 405 nm with a repeat reading after 30 minutes of incubation at 37[degrees]C. The concentrations of glutathione in the samples were determined based upon the linear equation of the line for the standard curve; the resultant concentrations were normalized to their respective cell count and reported as nM/10,000 cells.
Malondialdehyde (MDA) Assay: Cellular membrane damage was assessed by analyzing the levels of malondialdehyde bis diethyl acetal levels in the supernatant. This was accomplished by measuring thiobarbituric acid reactive substance (TBARS). Briefly, a standard curve using 1,1,3,3-tetraethoxypropane was prepared, and 0.1 mL of supernatant from the samples were added to respectively labeled 5 mL glass test tubes. Then, 0.5 mL of trichloroacetic acid (20% w/v) was added to all tubes and allowed to stand for 1 minute before adding 0.5 mL of thiobarbituric acid (0.67% in 0.2M NaOH). All tubes were then incubated in a 100[degrees]C shaking water bath for 45 minutes. They were then allowed to cool for 5 minutes prior to being centrifuged at 2500 rpm for 10 minutes. An aliquot of 0.1 mL was withdrawn from each standard and sample and placed in corresponding wells of a 96-well microtiter plate. The absorbances were then measured at a wavelength of 562 nm using a microtiter plate reader. The concentrations of MDA in the samples were determined based upon the linear equation of the line for the standard curve; the resultant concentrations were normalized to their respective cell count and reported as nM/10,000 cells.
Morphological Evaluation: The standard hematoxylin and eosin (H&E) procedure was used to demonstrate cellular morphology on the coverslips from columns 5 and 6. The coverslips were mounted on glass slides following the H&E staining procedure and subsequently were evaluated for general features (e.g., arrangement of cells), cytoplasmic features (e.g., size, shape, staining reaction), and nuclear features (e.g., size, shape, nucleoli). The slides were then digitized using Image-Pro Plus (Media Cybernetics, Inc.; Rockville, MD).
Statistics: Data analysis was performed using PASW Statistics (SPSS, Chicago) Version 18.0. Descriptive statistics generated included mean ([bar.y]) and standard error of the mean (SEM), represented in the form [bar.y] [+ or -] SEM. One-way ANOVA was performed for each experiment to determine any differences in response based on treatment. Multiple comparison procedures were done using Tukey's method when warranted by significant ANOVA results. All graphs were created using SlideWrite Plus (Advanced Graphics Software, Inc.; Encinitas, CA).
Cell Counts for M6P Study: Fibroblast cells treated with low (0.005 [micro]M) and medium (0.05 [micro]M) concentrations of M6P after 24 hours showed a trend toward increases in cell number. Treatment with high dose (0.5 [micro]M) M6P produced cell numbers that were more similar to control (Figure 1). ANOVA showed that none of the treatment groups represented a significant difference in cell count from control. After 48 and 72 hours of culturing the cells with M6P, cell numbers were suppressed when compared with control untreated cells. Low dose treatment at 48 and 72 hours produced a significantly lower cell count than control.
Cell Counts TGF-[beta]1 Study: After 24 hours, cells in the untreated control group showed significantly greater mitotic activity than cells in the medium (1200 ng/mL) and high (2000 ng/mL) groups. Mitotic activity for cells treated with low dose TGF-[beta]1 (200 ng/mL) was not significantly different from Control. At 48 hours, low dose treatment showed significantly greater mitotic activity than all other groups. By 72 hours, no significant differences were seen among the groups (Figure 2).
Choice of Doses and Time Point for Competitive Study: Since the 0.05 [micro]M dose of M6P was the lowest dose that did not show evidence of significantly decreasing mitotic activity or increasing cellular damage and the 200 ng dose of TGF-[beta]1, at 48 hours, showed the most significant increase in mitotic activity over Control, these doses and the 48 hour time point were chosen for the competitive study.
Cell Count for Competitive Study: The cell count for the group treated with TGF-[beta]1 alone revealed significantly greater mitotic activity than that of the control cells whereas the mitotic activity of the groups treated with M6P alone and with both TGF-Pi and M6P were not significantly different from the untreated control group (Figure 3).
GSH: The GSH assay revealed no significant difference between any of the three treatment groups and Control group (Figure 4).
MDA: The MDA assay showed that all three treatment groups had significantly lower cell membrane damage than the Control group (Figure 5).
Morphological Evaluation: Cells were evaluated 48 hours after treatment and were compared to the untreated control cells. Observations are reported in Table 1 and can be seen in Figure 6.
After an injury, the already tenuous blood supply of tendons is interrupted, creating a focal area of ischemia. Under such conditions, fibroblasts in the tendon have been shown to up-regulate the production of scar collagen. The collagen produced in such settings is not arranged in the typical parallel bundles seen in strong, healthy tendons. Instead, it forms a disorganized mesh that adheres to surrounding tissues, inhibiting the free gliding of the tendon. At the site of the scar, the tendon is never as strong as its pre-injury state because the fibers are not all aligned with the axis of force through the tendon. The overriding biological derangement responsible for this fibrotic response is hypoxia that precludes aerobic metabolism. In a hypoxic environment, hypoxia-inducible factor-1[alpha] (HIF- 1[alpha]) production is up-regulated in fibroblasts, inducing the cells to produce CTGF and TGF-[beta]. These two molecules interact with receptors on the fibroblasts to up-regulate the production of collagen. With this information in mind it is reasonable to examine HIF-1[alpha], CTGF, and TGF-[beta] as potential targets for inhibition of fibrosis.
Fibroblasts throughout the body have a receptor known as the M6P/IGF-II receptor, so named for two of its common ligands. An important additional ligand active at the M6P/IGF-II receptor is TGF-[beta]. TGF-[beta] interaction with the M6P/IGF-II receptor causes up-regulation of CTGF production by fibroblasts, resulting in increased production of collagen by the fibroblasts. Since both TGF-[beta]1 and M6P bind reversibly at the M6P/IGF-II receptor, M6P may competitively inhibit the binding of TGF-[beta]1. This would effectively inhibit the up-regulation of CTGF and collagen production by the fibroblasts.
Previous studies in our lab have shown that F6P but not M6P under hypoxic conditions is capable of decreasing HIF-1[alpha] and CTGF, suggesting M6P has a mechanism of action unrelated to isomerization to F6P. Studies in our lab on tendon healing have shown that M6P is capable of reducing scar tissue. This investigation has helped to provide the mechanism of action for M6P. In the experiments examining the effect of M6P on fibroblasts and its interaction with TGF-[beta]1, three things were determined: 1) M6P was not harmful to fibroblasts at either the medium or high doses of the concentrations tested; 2) TGF-[beta]1 significantly increased the mitotic activity of fibroblasts at the lowest of the concentrations tested; 3) the medium concentration of M6P was able to inhibit the action of the low dose of TGF-[beta]1 on fibroblast mitotic activity.
Overall, our results show that M6P is capable of reducing TGF-[beta]1-induced fibroblast proliferation and suggest that this is accomplished through competitive inhibition of the M6P/IGF-II receptor of fibroblasts.
The authors would like to acknowledge the support from the Department of Diagnostic and Clinical Health Sciences and the Department of Orthopedic Surgery and Rehabilitation at the University of Mississippi Medical Center.
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Gerri A. Wilson, David A. Black, Michelle A. Tucci, and Ham A. Benghuzzi
University of Mississippi Medical Center, Jackson, MS 39216
Table 1. Cellular Morphology at 48 Hours Control M6P 48 Hours General: numerous General: similar to singles and few loosely Control. cohesive aggregates. Cytoplasmic: similar to Cytoplasmic: few small, Control except exhibited round; numerous increased number of spindle-shaped with few spindle-shaped having extensions; few with multiple extensions and hydropic swelling having more with hydropic multiple extensions; swelling. scant with well-defined Nuclear: similar to borders. Control. Nuclear: basophilic; hyperchromatic; multiple micronucleoli. TGF-[beta]1 Both 48 Hours General: similar to General: similar to Control except numerous Control. tightly cohesive Cytoplasmic: similar to aggregates. Control. Cytoplasmic: similar to Nuclear: similar to Control except many Control. with round and irregular shapes and more extensions. Nuclear: similar to Control.
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|Author:||Wilson, Gerri A.; Black, David A.; Tucci, Michelle A.; Benghuzzi, Ham A.|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Apr 1, 2014|
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