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- Abstract: found
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Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy
research-article
Author(s):
M.P. Rangel
1 ,
V.K. de Sá
1 ,
V. Martins
1 ,
J.R.M. Martins
2
,
4 ,
E.R. Parra
1 ,
A. Mendes
2 ,
P.C. Andrade
1 ,
R.M. Reis
6
,
7
,
9 ,
A. Longatto-Filho
6
,
7
,
8
,
9 ,
C.Z. Oliveira
9 ,
T. Takagaki
3 ,
D.M. Carraro
5 ,
H.B. Nader
2 ,
V.L. Capelozzi
1
Publication date (Electronic):
08 May 2015
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.
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Most cited references47
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- Abstract: found
- Article: not found
Cancer associated fibroblasts: the dark side of the coin.
Paolo Cirri, Paola Chiarugi (2011)
Valid experimental evidence has recently shown that progression of malignant tumors does not depend exclusively on cell-autonomous properties of the cancer cells, but is also deeply influenced by tumor stroma reactivity and undergoes a strict microenvironmental control. Beside structural environmental components as extracellular matrix (ECM) or hypoxia, stromal cells as macrophages, endothelial cells, and cancer-associated fibroblasts (CAFs) play a definite role in cancer progression. This review summarizes our current knowledge on the role of CAFs in tumor progression towards an aggressive phenotype, with particular emphasis on invasiveness, stemness, and preparation of metastatic niche. The controversial origins of CAFs as well as the therapeutical implications of targeting CAFs for anticancer therapy are discussed.
- Record: found
- Abstract: found
- Article: found
The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering*
Cuixia Yang, Manlin Cao, Hua Liu … (2012)
Introduction The extracellular matrix glycosaminoglycan, hyaluronan (HA), 3 is an unbranched polymer composed of repeating glucuronic acid and N-acetyl glucosamine disaccharide units. The most widely distributed form of HA in normal tissues is native high molecular weight HA (called HMW-HA or nHA) with a high molecular weight (∼107 Da). The lower molecular weight forms of HA, also called hyaluronan oligosaccharides (oHA), are synthesized de novo or generated by either hyaluronidase-mediated degradation or oxidative hydrolysis of native HA under pathological conditions (1), including repair, inflammation, and tumor processes. nHA and oHA serve a variety of functions that are mediated by cell surface receptors, including cell motility, adhesion, migration, and metastasis (2–5). CD44 is a major cell surface receptor for HA, which has an N-terminal link module homology domain that is responsible for binding to HA. The interaction of CD44 and HA is strongly influenced by cell-specific factors (6), cell types (7), the state of CD44 activation (8), and the size of the HA ligand (9–11). Several mechanisms have been proposed for the regulation of receptor-mediated HA binding to CD44 (12–14). For example, the differences in HA chain length can influence both HA binding features and its functional consequences. High and low molecular weight forms of HA provoke distinct pro-inflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or anti-proliferative signals in appropriate cell types (15–19). Many studies showed that nHA exhibited anti-angiogenic and anti-inflammatory effects in several in vivo assays, inhibiting cell migration, proliferation, and sprout formation. In contrast, oHA stimulated cell proliferation and motility, exhibiting pro-inflammatory and pro-angiogenic effects in a variety of experimental systems (19–21). We previously reported that nHA and oHA regulated cell cycle progression through G1 phase in distinct manner in vascular endothelial cells (19). Although these reports demonstrate that the interaction of CD44 with oHA and nHA exerts distinct effects on cell biological behavior, the precise feature of the CD44 binding in response to different molecular weight HA remains obscure. In living cells the extracellular matrix including hyaluronan plays important role in the maintenance of appropriate cell-cell communication (22). Once the balance is disrupted, such as in tumor invasion, inflammation, or tissue remodeling, native high molecular weight HA is digested into small fragments (oHA). The loss of nHA and the appearance of its lower molecular weight forms could contribute to the changes of cell behavior and cell signaling. The most significant feature of HA is its highly repetitive nature. Therefore, the highly multivalent repeating disaccharide chains of HA interact with multiple, closely arrayed CD44 receptor molecules (23). However, the affinity of a single CD44-HA binding domain for HA is likely to be very low (10, 24, 25). Up to now, the molecular basis for functional distinction between the binding of CD44 with different sizes of HA is largely unclear. Clustering of CD44 is a typical character that affects cell response after stimulated by highly produced HA (26). In this study we utilized a fluorescence resonance energy transfer (FRET) technique to investigate the effects of oHA and nHA on CD44 clustering in COS-7 cells transfected with CD44 expression vectors. Furthermore, we chose HK-2 cells (human renal proximal tubule cells) and BT-549 cells (human breast cancer cell line) to evaluate the binding of oHA or endogenous nHA to CD44 because HK-2 and BT-549 cells naturally express abundantly CD44 and HA in naive status. We demonstrated that nHA induced CD44 clustering, which could be disrupted by oHA. These results provide direct evidence that high and low molecular weight forms of HA have distinct effects on CD44 clustering. EXPERIMENTAL PROCEDURES Reagents CD44 mAb (clone IM7, catalog no. 12-0441-81) for flow cytometry analysis was purchased from eBioscience. Biotinylated hyaluronan-binding protein was from Merck. Purified NA/LE mouse anti-CD44 (clone 515) was obtained from BD Biosciences Pharmingen. Anti-CD44 antibody (156-3C11), ERK inhibitor (U0126), and anti-phospho-ERK1/2 mAb were obtained from Cell Signal. nHA was obtained from Sigma. Hyaluronan oligosaccharides (oligomers) were prepared as described previously (27, 28), which was a mixed fraction of average molecular weight of 2.5 × 103 composed of 3–10 disaccharide units that were fractionated from testicular hyaluronidase (Sigma, type 1-S) digests of hyaluronan polymer (Sigma, sodium salt). 4-Methylumbelliferone and anti-vinculin mAb were purchased from Sigma. All other chemicals were of reagent grade or higher. Plasmid Construction The cDNA encoding the hematopoietic form of CD44 (CD44-H) was obtained by reverse transcription PCR (RT-PCR) using total RNA isolated from human peripheral blood lymphocytes and subcloned into the mammalian expression vectors pECFP-N1 and pEYFP-N1 (Clontech, Palo Alto, CA). The isoform CD44-H was used to denote a 80–90-kDa product (29). A plasmid encoding a positive control for the FRET experiment, PMT-CFP-YFP, was kindly provided by Dr. Thorsten Wohland (Department of Chemistry, National University of Singapore, Singapore) (30). In the positive control plasmid the plasma membrane target (PMT) sequence was fused to CFP at the N terminus, and the YFP gene was amplified and fused to CFP at the C terminus. Cell Culture and Transfection Mammalian cells COS-7 (simian virus 40-transformed African Green monkey kidney cell line), HK-2 cells (human renal proximal tubule cell line), and human breast cancer cells BT549 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. COS-7 cells were grown to 85% confluency and transfected by using Lipofectamine 2000 (Invitrogen). Binding of Exogenous HA to Transfected COS-7 Cells After transfection, COS-7 cells were incubated with 40 μg/ml fl-HA (Merck) for 1 h at 4 °C as described previously (29), then the cells were treated by EDTA and washed three times in phosphate-buffered saline (PBS). After fixation with 1% paraformaldehyde and nuclear counterstaining by propidium iodide, plasma membrane-bound fl-HA was observed under a confocal microscope (Nikon A1, Tokyo, Japan) and analyzed using a flow cytometer (Beckman-Coulter, Brea, CA). Flow Cytometric Analysis Cultured cells were harvested and washed with washing buffer (PBS supplemented with 2% bovine serum albumin, pH 7.4). A single cell suspension (106/ml) was incubated with specific antibodies or isotope control antibodies on ice for 1 h. Cells were washed 3 times with washing buffer, and 1 μl of the specific antibody was added for 1 h on ice. Cells were analyzed in a flow cytometer (Beckman-Coulter). At least 6000 cells were analyzed per sample in all experiments. All experiments were performed at least twice. FRET Measurements with Spectroscopy FRET spectroscopy was performed essentially as described previously (31). Briefly, COS-7 cells were plated into 96-well black plates for 24 h then transfected with plasmids encoding CD44-YFP and CD44-CFP, PMT-CFP-YFP, or co-transfected with CD44-YFP and CD44-CFP. For FRET measurements, fluorescence intensities of CFP, YFP, and PMT-CFP-YFP were measured using a microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT). After an excitation narrowed at 440 nm, the special excitation channel of CFP, emission fluorescence was obtained at 485 ± 10 nm (CFP emission peak) and 528 ± 10 nm (YFP emission peak). If FRET occurs, the fluorescence at 528 nm will be enhanced for efficient energy transfer between donor and acceptor. A modified method for FRET evaluation was applied according to a previous report (31). Calculation of the ratio between emission at 528 and 485 nm was used as an indicator of FRET efficiency. Higher values of 528/485-nm ratio indicated higher efficiency of FRET values. Fluorescence from non-transfected cells and background scattering light brought by the equipment was subtracted from each sample to obtain the specific fluorescence. FRET-based Dose-response Experiment for Competitive Inhibition of oHA A primary dose-response experiment with varying concentrations of oHA was performed using a microplate fluorescence reader (Bio-Tek Instruments). Cells co-transfected with CD44-YFP and CD44-CFP were pretreated with nHA, then incubated with different doses of oHA (0, 31.25, 62.5, 125, and 250 μg/ml). The fluorescence was recorded in monochromatic mode with excitation at 440 nm and emission at 485 and 528 nm. The FRET efficiency, the ratio between emission at 528 and 485 nm, was used as an indicator of the dose-dependent changes in HA treatments. FRET Imaging FRET imaging was performed basically as described before (32, 33) on a confocal laser scanning microscopy (Nikon A1, Tokyo, Japan). For sensitized fluorescence emission measurements, images were acquired sequentially through YFP, CFP, and FRET channels. The microscopy sets used were YFP (excitation, 500/25 nm; emission, 526–568 nm), CFP (excitation, 458 nm; emission, 461–504 nm), and FRET (excitation, 458 nm; emission, 526–568 nm). FRET efficiency was determined using NIS-Elements AR3.1 Imaging software (Nikon, Tokyo, Japan). Fluorescence through the FRET channel consisted of a FRET component (“corrected” FRET, FRET) and non-FRET components, including spectral bleed through and cross-excited fluorescence. The non-FRET components were subtracted as described previously (34). FRETC was calculated on a pixel-by-pixel basis for the entire ”region of interest” image, where FRET, CFP, and YFP were presented as background-subtracted fluorescence intensities acquired through the FRET, CFP, and YFP channels, respectively. In our experimental conditions we used the following equation: FRETC = FRET − 0.55 × CFP − 0.11 × YFP. The 0.55 and 0.11 were the fractions of bleed-through of CFP and YFP fluorescence to the FRET channel, respectively. The fractions were calculated by measuring fluorescence intensities of cells individually expressing CD44-CFP or CD44-YFP through the CFP, YFP, and FRET channels. All the experiments were performed at room temperature. Acceptor-photobleaching-based FRET Measurement Confocalmicroscopy imaging and photobleaching experiments were performed at room temperature using an Nikon A1 laser scanning confocal microscopy system. If any interactions leading to energy transfer were present in the cells, photobleaching of the acceptor would lead to an increase in donor fluorescence because it would no longer be quenched by the acceptor. In the photobleaching experiments, a 60-s pulse of high intensity laser at 514 nm (100% exposure at 514 nm beam) was applied to locally bleach the YFP signal acceptor in a whole cell. Data acquisition was performed in the sequential line mode for the best spatiotemporal reliability. The sample area was scanned at the resolution of 1024 × 1024 pixels. The CFP and YFP were excited at 458 and 514 nm, respectively. Two frames of pre-bleaching and post-bleaching CFP (donor) fluorescence intensities were recorded. The FRET efficiency (E) was calculated by E = 1 − (F pre/F post)·F pre, where F pre and F post indicate the fluorescence intensity of the donor before and after acceptor photobleaching, respectively. Immunocytochemistry Analysis of CD44 Clustering The clustering of CD44 was analyzed by immunocytochemistry. HK-2 and BT-549 cells were cultured on glass coverslips, then incubated with DMEM (control), oHA, hyaluronidase, and 4-methylumbelliferone (4-MU). After incubation, cells were washed briefly with PBS, fixated in 4% paraformaldehyde for 10 min, then permeabilized using Triton X-100 (0.1% v/v in PBS) for 10 min at room temperature. The cells were incubated with anti-CD44 antibody (156–3C11) overnight at 4 °C, then stained with an Alexa Fluor 488-conjugated chicken anti-mouse IgG antibody (Invitrogen) for 1 h in the dark at room temperature. The coverslips were then placed onto slides using glycerol as a mountant and visualized under confocal microscopy (Nikon A1, Tokyo, Japan). Quantitation of Endogenous Hyaluronan Generation To understand the effect of oHA on endogenous hyaluronan generation, the amount of liberated and cell-associated hyaluronan was determined as described previously (35). Triplicate cultures of cells were seeded in a 6-well culture plate and grown for 24 h to 85% confluence. Cells were treated with different doses of oHA (0, 31.25, 62.5, 125, and 250 μg/ml) for 2 h then harvested by trypsinization and counted using a Coulter counter. Media were used for quantitation of the liberated HA. Cell-associated extracellular HA was obtained by centrifugation of the cell/trypsin fraction at 2000 rpm in a Beckman TJ-6 centrifuge where the supernatant was quantitated for HA. The extracellular liberated and cell-associated fractions were pooled and characterized for HA concentration with the 125I-labeled HA radioimmunoassay, the detection limits of which is >5.5 ng/ml. Chemical Cross-linking for CD44 Association at the Cell Surface Cells were treated with DMEM, oHA, testicular hyaluronidase or 4-MU, then the cells were washed 5 times with ice-cold PBS (20 mm sodium phosphate, 0.15 m NaCl, pH 8.0). CD44 cross-linking was performed by incubation with 2 mm bis(sulfosuccinimidyl) suberate (BS3) (Pierce) for 1 h at 4 °C and quenched by incubation with 20 mm Tris, pH 7.5, for 15 min at room temperature. Cells were washed twice with PBS and lysed with cell lysis buffer. Western Blotting Cells were harvested and homogenized in ice-cold sodium dodecylsulfate (SDS) lysis buffer. Total cell lysates were collected, and equal quantities of protein were separated by SDS-PAGE and blotted onto a PVDF membrane. The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h, washed for 1 h in TBS, and incubated with CD44 (156–3C11, Cell Signal) or phospho-ERK1/2 mAb (Cell Signal) at 4 °C for 3 h. Then the membranes were washed with 1× Tris-buffered saline/Tween 20 (TBS/T) buffer for three times (5 min each time) and incubated with HRP-conjugated polyclonal secondary antibody for 1 h. The membranes were developed with the enhanced plus chemiluminescence assay (Pierce) according to the manufacturer's instructions. Cell Adhesion Assay The adhesion of cells was examined as previously described (36, 37). After being pretreated with control (DMEM), oHA, and nHA for 2 h, cells were detached with 5 mm PBS/EDTA. Cells (5 × 103) in spreading medium (DMEM with 0.1% heat-inactivated FBS) were seeded onto a 96-well plate. After 30 min at 37 °C, unbound cells were removed, and the remaining cells were counted. Treatment with ERK inhibitor (U0126; 5 μm, 24 h) was performed to detect the effect of inhibition of ERK on cell adhesive ability. Three independent experiments were performed. Statistical Analysis The data were presented as the mean ± S.D. All statistical analysis was performed using SPSS11.0. The unpaired Student's t test was used for the comparisons between two different groups. p less than 0.05 was considered significant. RESULTS Characterization of CD44-YFP and CD44-CFP Constructs COS-7 cells were chosen to characterize CD44-CFP and CD44-YFP constructs due to the absence of endogenous expression of CD44 and hyaluronan in these cells (Fig. 1 A, first lane, and supplemental Figs. S1). Western blot analysis revealed a band at ∼85 kDa in pCD44H-transfected COS-7 cells, corresponding to the molecular weight of the standard form of human CD44 (Fig. 1 A, second and third lanes). Flow cytometry analysis indicated that CD44 was successfully translocated onto the cell surface (Fig. 1 B). As the negative control, no CD44 expression was detected in COS-7 cells transfected with mock vector (Fig. 1 B). FIGURE 1. Binding of exogenous fl-HA by COS-7 cells transfected with CD44 expression constructs. 24 h post-transfection, COS-7 cells were analyzed by Western blot and flow cytometry analysis. The capacity of transfected cells to bind exogenous fl-HA was analyzed by flow cytometry and immunocytochemistry analysis. A, shown is immunoblotting of CD44 in the lysates from CD44-CFP (lane 2)- and CD44-YFP (lane 3)-transfected COS-7 cells. Naive COS-7 cell was used as the negative control. B, cell surface expression of CD44 in CD44-CFP- and CD44-YFP-transfected COS-7 cells was assessed by flow cytometry. Histograms indicate log fluorescence intensity (x axis) versus relative cell number (y axis). 1, naïve COS-7 cells was used as mock; 2, isotype-matched antibody as negative control; 3, pECFP-N1 transfected cells; 4, pEYFP-N1 transfected cells; 5, CD44-CFP transfected cells; 6, CD44-YFP transfected cells. C, shown is binding and uptake of exogenous fl-HA by transfected COS-7 cells. 1, naïve COS-7 cells was used as negative control; 2, CD44-CFP transfected COS-7 cells; 3, CD44-YFP transfected COS-7 cells. D, binding of exogenous fl-HA by transfected COS-7 cells was detected by immunocytochemistry analysis. Nuclei were visualized by propidium iodide (PI) staining. COS-7 cells were transfected with CD44-CFP or CD44-YFP respectively. Naïve COS-7 cells were used as mock control. After transfection, captured fl-HA (green fluorescence) was visualized at the surface of CD44-transfected COS-7 cells by flow cytometry and confocal microscopy analysis (Fig. 1, C and D). As negative control, untransfected cells showed no capacity to bind fl-HA (Fig. 1 D). nHA and oHA Regulate CD44 Clustering in Distinct Manner To evaluate the effects of nHA and oHA on CD44 clustering, we performed FRET. As shown in Fig. 2 A. CD44-CFP emitted mainly at 485 nm and with lower intensity at 528 nm, although CD44-YFP showed emission mainly at 528 nm. Co-transfection of CD44-CFP and CD44-YFP initiated two separate emission intensities. We observed typical FRETs between CFP and YFP in positive control tandem PMT-CFP-YFP construct with no FRET in two negative control pairs CD44-CFP with EYFP-N1 and ECFP-N1 with CD44-YFP (Fig. 2 A). FIGURE 2. nHA and oHA regulate CD44 clustering in COS-7 cells transfected with CD44 expression constructs. Clustering of CD44-CFP and CD44-YFP was detected by the efficiency of FRET between donor (CFP) and acceptor (YFP). Fluorescence at 485 nm (CFP emission peak) and 528 nm (YFP emission peak) were measured after excitation only at 440 nm (CFP special excitation channel). The ratio of fluorescence at 528 and 485 was estimated to indicate the transferred energy of CFP to YFP. CD44-CFP, transfected with CD44-CFP plasmid; CD44-YFP, transfected with CD44-YFP; CFP, transfected with pECFP-N1 vector; YFP, transfected with pEYFP-N1 vector; PMT-CFP-YFP, CFP-YFP fusion protein with a plasma membrane target (PMT) sequence at the CFP N terminus. A, fluorescence spectra of recombinant CFP and YFP proteins are shown. B, nHA and oHA treatment led to the changes in the efficiency of energy transfer from cyan to yellow fluorescent proteins in COS-7 cells co-transfected with CD44-CFP and CD44-YFP. C, oHA abrogated CD44 clustering triggered by exogenous nHA. After exposed to nHA, cells were treated with oHA of different concentrations (0–250 μg/ml) for another 2 h. The fluorescence intensity was plotted as the mean (n = 8) ±S.D. RFU, relative fluorescence units. The ratio of fluorescence between 528 and 485 nm was used to estimate energy transfer efficiencies. As shown in Fig. 2 A, the highest ratio was obtained with PMT-CFP-YFP (∼5.77). Co-expression of CD44-CFP and CD44-YFP without HA treatment showed a low ratio at 1.44, indicating the weak clustering of CD44 in transfected COS-7 cells (Fig. 2 A). When COS-7 cells co-transfected with CD44-CFP and CD44-YFP were treated with nHA, the 528/485-nm ratio increased to 4.7. Nevertheless, when cells were treated with oHA, the 528/485-nm ratio decreased to 1.38 (Fig. 2 B). The decrease of about 70% in the 528/485-nm ratio in oHA-treated cells is probably due to the displacement of nHA by oHA. Moreover, the primary dose dependence experiment indicated that the reduction of FRET signal by oHA was found to be concentration-dependent (Fig. 2 C), reaching a plateau at 125 μg/ml, which was the minimum concentration that could destroy maximally the CD44 clustering induced by nHA (Fig. 2 C) and thereby be selected for the following experiments. Next, FRET Imaging was applied to evaluate the clustering of CD44 in response to nHA or oHA in living cells. COS-7 cells were co-transfected with CD44-CFP and CD44-YFP constructs. As shown in Fig. 3, no FRET signal was detected in resting cells in the blank group and oHA treatment group. However, nHA exposure generated FRET signal within 2 h, indicating the clustering of CD44-CFP with CD44-YFP. Such FRET signal was significantly reduced when the cells were treated with oHA (Fig. 3). Moreover, anti-CD44 mAb induced a very weak FRET signal (Fig. 3). Taken together, these data suggest that nHA induces CD44 clustering, although oHA inhibits CD44 clustering. FIGURE 3. FRET analysis of CD44-CFP/CD44-YFP clustering in COS-7 cells. COS-7 cells were co-transfected with CD44-CFP and CD44-YFP, and treated with medium, nHA (125 μg/ml), oHA (125 μg/ml), or anti-CD44 mAb (10 μg/ml) for 2 h. After exposed to nHA, one group of cells was treated with oHA (125 μg/ml) for another 2 h. Cells treated with culture medium were used as negative control. Another three controls were used: two negative control pairs CD44-CFP with EYFP-N1 and ECFP-N1 with CD44-YFP and a positive control tandem PMT-CFP-YFP construct. nHA induced CD44-CFP-CD44-YFP clustering as indicated by the FRET signal. oHA did not induce CD44-CFP-CD44-YFP clustering but reduced FRET signal induced by nHA. Similar findings were observed when the cells were treated by anti-CD44 mAb. FRET images were normalized with respect to FRET efficiency and shown in a pseudocolor mode. The scale indicated FRET efficiency from purple (0) to red (90%); scale bar = 100 μm. The results represent five separate experiments; more than three cells were analyzed each time. Furthermore, immunocytochemistry analysis of CD44-CFP/CD44-YFP clustering in COS-7 cells revealed that CD44 was distributed in clustering form in nHA-treated cells, although oHA reduced the number of cells showing CD44 clusters (Fig. 4 A). In addition, a protein chemical cross-linking experiment was used to assess CD44 clustering. Western blotting analysis showed the additional CD44 bands in CD44-transfected COS-7 cells after nHA incubation and BS3 treatment, indicating that cross-linking had occurred between CD44 protein. Likewise, oHA reduced the amount of cross-linked CD44 with the obvious decrease in CD44 dimers (Fig. 4 B). FIGURE 4. Immunocytochemistry and chemical cross-linker analysis of CD44 clustering in transfected COS-7 cells. A, clustering of CD44 with different treatments was analyzed by immunocytochemistry. CD44-transfected COS-7 cells were pretreated with DMEM (Control), oHA (125 μg/ml), or nHA (125 μg/ml) for 2 h at 37 °C. After exposure to nHA, one group of cells was treated with oHA (125 μg/ml) for another 2 h. CD44 was distributed into clusters in nHA-treated cells (indicated by the arrows). B, CD44-transfected COS-7 cells were pretreated with DMEM (Control), oHA (125 μg/ml), and nHA (125 μg/ml) for 2 h at 37 °C. After exposure to nHA, one group of cells was treated with oHA (125 μg/ml) for another 2 h, then all cells were incubated with or without BS3 protein cross-linker (2 mm) for 1 h at 4 °C. The cross-linked CD44 on cell surface was detected by Western blot analysis. Without BS3 treatment, immunoblotting of the cell lysate revealed a major 85-kDa band. Incubation of cells with BS3 led to the appearance of additional immunoreactive bands at 170 kDa. Shown are representative images from three independent experiments with similar results. CD44 Clustering in COS-7 Cells Detected by Photobleaching FRET FRET measured by donor dequenching after acceptor photobleaching was utilized to detect CD44 clustering. COS-7 cells were cotransfected with CD44-CFP and CD44-YFP plasmids and treated with 125 μg/ml nHA. The intensity of CFP fluorescence was increased, as the CD44-YFP was photobleached (Fig. 5 A). However, for other cells that were not photobleached within the same field of view, the intensity of both CFP and YFP did not change obviously (Fig. 5 A). FIGURE 5. Photobleaching FRET analysis of CD44-CFP/CD44-YFP clustering in COS-7 cells. FRET was measured by donor dequenching after acceptor photobleaching in COS-7 cells co-transfected with CD44-CFP and CD44-YFP. Two frames before and after the bleaching interval were presented. Bleached region of YFP was shown (arrows). A, images were acquired 2 h after the cells were treated with 125 μg/ml nHA. B, images were acquired 2 h after the cells were treated with 125 μg/ml nHA followed by treatment with 125 μg/ml oHA for 2 h. C, the bar graphs representing FRET efficiency (E) were from five independent experiments performed as shown in A. The efficiency was determined by the acceptor photobleaching method and was measured only in the acceptor bleached area. Cells outside the bleached region were used as controls. D, bar graphs representing FRET efficiency (E) were from the five independent experiments performed as shown in B. All data are represented as the mean ± S.D. *, p < 0.001. When the cells treated with 125 μg/ml nHA were further treated by 125 μg/ml oHA after the YFP molecule was photobleached, the intensity of CD44-CFP showed no significant increase (Fig. 5, B and D). The average energy transfer efficiency for the cells with photobleached acceptors in the nHA-treated cells was 51%, compared with −1.0% in the controls (Fig. 5 C). After oHA treatment, the energy transfer efficiency was decreased to −15.3% (Fig. 5, B and D). These results confirm that nHA induces CD44 clustering, although oHA inhibits CD44 clustering. Endogenous nHA-induced CD44 Clustering Is Disrupted by Hyaluronan Oligosaccharides To confirm the opposite effects of nHA and oHA on CD44 clustering, additional experiments were performed in HK-2 cells (human renal proximal tubule cells) and BT-549 cells (human breast cancer cells), which unlike COS-7 cells without expression of HA and CD44, exhibiting abundant expression of CD44 as showed in Fig. 7 and supplemental Figs. S2. In addition, the native HA was highly expressed and organized into cable-like structures in both cells (Fig. 6 A). Confocal microscopy revealed that CD44 was distributed in clustering form in naïve HK-2 and BT-549 cells (Fig. 6 B). To be sure that the CD44 clusters are induced by endogenous HA, the HA synthesized inhibitor, 4-MU, was used to inhibit HA secretion before CD44 clusters were determined. Our results indicated that cells treated with 0.5 mm 4-MU for 24 h showed a substantial inhibition of HA staining (Fig. 6 A) and CD44 clustering (Fig. 6 B), demonstrating that endogenous HA is the indispensable factor of the formation of CD44 clusters. Similar findings were observed when the cells were treated with bovine testicular hyaluronidase, which was used as positive control. FIGURE 6. oHA abrogates CD44 clustering triggered by endogenous nHA in HK-2 and BT-549 cells. A, the expression of hyaluronan with or without oHA treatment was detected by immunocytochemistry analysis. Endogenous nHA was cross-linked into cables (indicated by the arrows). Sections were imaged by microscopy (×20 objective). To confirm the nature of HA staining, cells were treated with bovine testicular hyaluronidase (250 μg/ml) at 37 °C for 2 h before fixation and the addition of biotinylated HA-binding protein. 4-MU (0.5 mm, 24 h), the hyaluronan synthesis inhibitor, was used as positive control. B, the clustering of CD44 with or without oHA treatment was analyzed by immunocytochemistry. CD44 was distributed into clusters in naïve HK-2 and BT-549 cells (indicated by the arrows). As a positive control, cells were treated with bovine testicular hyaluronidase (250 μg/ml) at 37 °C for 2 h. Treatment with 4-MU (0.5 mm, 24 h) was performed to detect the effect of inhibition of endogenous HA synthesis on CD44 clustering. In addition, after treatment with oHA, the amount of cell-associated HA on cell surface was significantly reduced (Fig. 6 A). Accordingly, the number of cells showing CD44 clusters was reduced, although CD44 expression on cell surface was not significantly changed after oHA treatment (supplemental Figs. S2). Interestingly, oHA treatment had hardly any inhibitory effect on endogenous HA generation (Table 1), suggesting that the reduced clusters of CD44, mediated by oHA, was due to direct competition with the same binding site on CD44. TABLE 1 oHA treatment has no effect on the amount of endogenous HA generation Cells Quantitation of hyaluronan production after oHA treatment for 2 h Control (DMEM) 62.5 μg/ml oHA 125 μg/ml oHA 250 μg/ml oHA fg HK-2 685.0 ± 14 687.0 ± 27 689.0 ± 31 680.0 ± 24 BT-549 997.0 ± 36 995.0 ± 25 1015.0 ± 45 945.0 ± 42 Furthermore, protein chemical cross-linking experiment was used to assess CD44 clustering on cell surface of HK-2 and BT-549 cells. CD44s had an apparent molecular mass of ∼85 kDa without BS3 treatment (Fig. 7), and the addition of oHA or hyaluronidase did not change the apparent molecular weight of CD44s (Fig. 7, A and B). However, after subsequently BS3 treatment, a new band of CD44 emerged corresponding to apparent molecular mass products of 170 kDa, indicating that CD44 dimers formed after protein cross-linking (Fig. 7 A). To confirm the role of endogenous HA synthesis in CD44 clustering, 4-MU was used to inhibit HA secretion before incubation with or without BS3 protein cross-linker. The results indicated that treatment with 4-MU inhibited the formation of CD44 dimers (Fig. 7, C and D), providing other evidence that endogenous HA is the most critical factor in the formation of CD44 clusters. Next, the clustering of CD44 in response to oHA treatment was detected to evaluate the opposite effects of nHA and oHA. As expected, oHA treatment significantly reduced the amount of CD44 dimers (Fig. 7, A and B), further confirming that oHA could attenuate CD44 clustering on the cell surface stimulated by endogenous nHA. An obvious decrease in CD44 dimers was also observed in cells treated with bovine testicular hyaluronidase (Fig. 7, A and B), which was used as the positive control for the disruption of endogenous nHA. FIGURE 7. CD44 clustering on the surface of HK-2 and BT-549 cells was assessed by chemical cross-linker analysis. A and B, HK-2 cells (A) and BT-549 cells (B) were pretreated with DMEM (Control), oHA (250 μg/ml), and hyaluronidase (250 μg/ml) for 2 h at 37 °C, then incubated with or without BS3 protein cross-linker (2 mm) for 1 h at 4 °C. Treatment with bovine testicular hyaluronidase (250 μg/ml) was used as positive control. After incubation, the cross-linking reaction was quenched by 20 mm Tris, pH 7.5. Cells were washed twice and lysed with cell lysis buffer. The cross-linked CD44 on the cell surface was detected by Western blot analysis. Without BS3 treatment, immunoblotting of cell lysate revealed a major 85-kDa band. Incubation of cells with BS3 led to the appearance of additional immunoreactive bands at 170 kDa. C and D, to confirm the effect of endogenous HA on CD44 clustering, HK-2 (C) cells and BT-549 cells (D) were pretreated with or without the hyaluronan synthesis inhibitor 4-MU (0.5 mm, 24 h) then incubated with or without BS3 protein cross-linker (2 mm) for 1 h at 4 °C. The cross-linked CD44 on cell surface was detected by Western blot analysis. Shown are representative images from three independent experiments with similar results. oHA and nHA Induces Different Cell Adhesive Abilities Having demonstrated the different effects of oHA and nHA on CD44 clustering, we next wondered whether oHA and nHA exert distinct effects on cell biological behaviors. To address this, we examined the adhesion of CD44-transfected COS-7 cells to 96-well plates. The results showed that oHA promoted the attachment of cells to the 96-well plate (Fig. 8 A). In contrast, nHA showed no effects on the attachment (Fig. 8 A). To determine if endogenous HA could influence the cell binding, a radioimmunoassay was performed to observe the effects of oHA on HA generation. Our results showed that oHA did not affect endogenous HA synthesis as long as 0 ∼ 2 h (supplemental Table S1). As the cells were attached onto the plate only for 30 min, the increase of adhesive ability by oHA treatment may be due to the regulation of other adhesion factors by oHA, such as F-actin or focal adhesions, etc., leading to cells attached to the polystyrene plate. FIGURE 8. Effects of nHA and oHA on cell adhesion. A, CD44-transfected COS-7 cells (5 × 103) were seeded in 96-well plates after pretreated with DMEM (control) or soluble oHA or nHA. After 30 min at 37 °C, the bound cells were counted. The results represented the average of three independent experiments in triplicate and are shown as the mean ± S.D. *, p < 0.05. B, effects of HA on actin fiber and focal adhesions are shown. CD44-transfected COS-7 cells treated with DMEM (control), oHA (125 μg/ml), and nHA (125 μg/ml) were fixed, permeabilized, and then stained using FITC-labeled phalloidin to visualize actin filaments. Cells treated with oHA had more intense F-actin stress fibers (arrows). The cells also were stained with anti-vinculin for focal adhesion staining, and the nuclei were stained using DAPI. The signals against vinculin and DAPI were superimposed. Cells treated with oHA showed the increased quantity of vinculin-positive adhesion plaques in the undersurface of cells (arrows). Note that both F-actin and vinculin are co-distributed in focal adhesions. Stress fibers and associated focal adhesions in cells constitute a contractile apparatus that regulates cell motility and contraction, and specific cytoskeletal rearrangements of cells have been implicated in cell adhesion and migration (37, 38). To characterize the effects of oHA and nHA on the microfilament organization and focal adhesion contact formation, fluorescence cytostaining using FITC-labeled phalloidin and immunostaining of vinculin were investigated by confocal microscopy. As illustrated in Fig. 8 B, staining with phalloidin for cytoskeletal architecture showed that the cells treated with oHA had more intense F-actin stress fibers arranged in spike-like protrusions resembling microvilli-like structures (Fig. 8 B). In contrast, soluble nHA showed no effects on the cell actin reorganization in CD44-transfected COS-7 cells (Fig. 8 B). Moreover, vinculin-specific immunofluorescence assay demonstrated that oHA treatment induced an increased number and size of adhesion plaques under the cells, whereas nHA had no effects (Fig. 8 B). These results are consistent with previous studies reporting that HA chains of different sizes affect F-actin organization and focal contact formation, resulting in changes of cell adhesion (39, 40). To further understand the intracellular mechanism through which oHA enhances cell adhesion, we next studied one of the important cellular signal pathways, ERK1/2, which has been regarded as a crucial regulator of cell adhesion (41) and also has been reported to interact with CD44 directly (42, 43). Western blot analysis revealed that phospho-ERK-1/2 was significantly increased after oHA treatment compared with untreated cells (Fig. 9, A and B). However, after nHA treatment, phosphorylation levels of ERK1/2 were not obviously changed (Fig. 9, A and B). Additionally, the up-regulated level of pERK1/2 by oHA was decreased after the blockage of CD44 by anti-CD44 mAb (Fig. 9, C and D). To further identify the role of ERK involved in cell adhesion, a blocking of ERK experiment was performed. As a result, the addition of the specific ERK inhibitor strongly decreased both fundamental and oHA-induced cell adhesion capacity (Fig. 10), suggesting that pERK1/2, originated from CD44/HA interactions, functions as a downstream signal regulating cell adhesion. FIGURE 9. oHA stimulates ERK signaling in HK-2 and BT-549 cells. A and B, shown is the effect of HA on ERK1/2 phosphorylation. HK-2 (A) and BT-549 (B) cells were treated with DMEM (Control) and nHA or oHA for 30 min, and phosphorylated ERK1/2 (pERK1/2) was detected by Western blot analysis. C and D, HK-2 (C) and BT-549 (D) cells were pretreated with or without anti-CD44 mAb (50 μg/ml) for 12 h and then treated with oHA (125 μg/ml) for 5 min. Cells treated with DMEM were used as control. Normal mouse IgG (NIgG) was used as negative control. The phosphorylated ERK1/2 (pERK1/2) was detected by Western blot analysis. Shown are representative blots from three independent experiments with similar results. The band intensities were analyzed by densitometry analysis. *, p < 0.05 compared with the controls. FIGURE 10. Effect of ERK inhibitor (ERKi) on CD44-dependent cell adhesion. A, HK-2 cells, pretreated with ERK inhibitor (5 μm) for 24 h, were incubated with or without oHA (125 μg/ml) for 2 h then harvested and allowed to attach (5000 cells) for 30 min on 96-well plates. B, shown is the effect of ERK inhibitor on HK-2 cell ERK1/2 phosphorylation. HK-2 cells treated with ERK inhibitor for 24 h were harvested, and equal amounts of cell extract protein were separated by PAGE and blotted with phosphorylated ERK1/2 (pERK1/2) utilizing specific antibodies. Densitometry analysis of the bands was normalized against GAPDH and plotted. C, BT-549 cells, pretreated with ERK inhibitor (5 μm) for 24 h, were incubated with or without oHA (125 μg/ml) for 2 h. The cells were then harvested and allowed to attach (5000 cells) for 30 min on 96-well plates. D, shown is the effect of ERK inhibitor on BT-549 cell ERK1/2 phosphorylation. BT-549 cells were treated with ERK inhibitor for 24 h, and the phosphorylated ERK1/2 (pERK1/2) was detected by Western blot analysis. Densitometry analysis of the bands was normalized against GAPDH and plotted. All results represent the average of three separate experiments. The means ± S.E. are plotted; statistical significance is as follows: *, p < 0.05 compared with the respective control samples. DISCUSSION It has been reported that the biological effect of HA depends on its molecular weight (44–46). Specifically, CD44 binding of a fraction of HA defined operationally as “low molecular weight” (4–20-mer) has been reported to stimulate cell proliferation (18), whereas higher molecular weight fractions exert an inhibitory effect (44). In this study we analyzed the influence of high and low molecular weight hyaluronan on CD44 clustering. Our results provide direct evidence for the distinct features of the interactions between CD44 and different size forms of HA. FRET analysis showed that nHA induced a drastic increase of FRETc signal, which indicated an augment of cell membrane surface CD44 aggregation. However, the addition of oHA caused an attenuation of such aggregation. These results suggest that oHA abrogates CD44 clustering induced by exogenous native hyaluronan. Considering that both oHA and nHA have the same binding site on CD44, the abolishment of CD44 clustering could be due to the displacement of nHA by oHA. Indeed, previous studies reported that the hyaluronan around the cells was displaced by oligosaccharides (47, 48). HA is known to contain multiple (up to a few thousand) CD44 binding sites due to the repetitive structure (GlcNAcβ1–4GlcUA) n and could interact simultaneously with many CD44 receptors on the cell surface (49). Moreover, by reconstituting CD44 expression in AKR1 cells that lack endogenous CD44 expression, Lesley et al. (21) showed that HA binding at the cell surface is a complex interplay of multivalent binding events affected by the size of the multivalent HA ligand and the quantity and density of CD44 at the cell surface. In this study CD44 clustering induced by HA was further investigated in HK-2 and BT-549 cells, which naturally express abundant CD44 and endogenous native HA. Our immunocytochemistry results revealed that excessively secreted native HA was cross-linked into cables, and CD44 was distributed into clusters in HK-2 and BT-549 cells. Furthermore, treatment with 4-MU, an inhibitor of hyaluronan synthesis, diminished the hyaluronan around the cells and CD44 clusters on the cell surface, indicating that the cluster of CD44 in HK-2 and BT-549 cells is dependent on endogenous hyaluronan synthesis. In addition, protein chemical cross-linking experiment provided further data that inhibition of endogenous HA synthesis by 4-MU obviously inhibited the formation of CD44 clusters, suggesting that CD44 clustering on the cell surface was mainly mediated by endogenous nHA in HK-2 and BT-549 cells. Therefore, our data complement and extend previous findings by demonstrating the multivalent binding between HA and CD44 in cells that endogenously express these molecules. Although by an artificial supported lipid bilayer membrane system, the multivalent interactions between multiple binding sites on the HA chain and multiple CD44 receptors on the surface were analyzed quantitatively through the presentation of CD44 at controlled density (23), our approach differs from previously reported binding assays in that we analyzed the multivalent binding of HA and CD44 in living cells. The long chains of HA possess multivalent or repeated sites for CD44 binding, whereas hyaluronan oligomers contain only one to two binding sites of CD44 (21, 23). By FRET technique, we found that oHA strongly reduced CD44 clustering induced by exogenous native hyaluronan. Moreover, although treatment of HK-2 and BT-549 cells with oHA had no influence on the amount of CD44 on cell surface and the endogenous HA generation, oHA significantly reduced the number of cells showing CD44 clusters. These results suggest that oHA regulates CD44 clustering in an opposite manner as nHA by replacing the multivalent HA-CD44 interaction with a monovalent interaction, leading to the disruption of CD44 clustering. CD44 is a member of the hyaluronan receptor family and serves as an adhesion molecule that binds ligand of the extracellular matrix. Therefore, CD44 is usually reported to mediate cell-cell and cell-matrix adhesion, including lymphocyte homing, hemopoiesis, and cell migration (6, 50, 51). To investigate the biological significance of CD44 clustering induced by HA, we examined the adhesive ability of CD44-transfected cells. Our results demonstrate that HA specifically regulates cell adhesion in a manner dependent on molecular weight. That is, oHA promoted the attachment of cells to the polystyrene plate, whereas nHA showed no effects under the same condition. This is consistent with the previous report (37). However, there were some reports indicating that oHA could regulate endogenous HA generation (52). To determine if the cell adhesion was mediated by endogenous HA secretion, we further investigate the effect of oHA on cell HA production. As shown in the results, oHA showed no effects on HA generation throughout the experiments. Interestingly, previous studies reported that oHA mainly restrained the endogenous HA synthesis (28), whereas our data showed no inhibition. This controversial result needs a future investigation. Together with the result that oHA promoted cell adhesion, our study suggested that the cell adhesion may be mediated by CD44-HA interactions but not the endogenous HA. Scientists from other laboratories have demonstrated the role of CD44 in cell motility in its ability to interact with cell skeletal protein, such as actin microfilaments and adhesion molecules (53–55). Cell adhesion requires organized actin stress fibers and focal adhesion contacts (56). To character the functional consequences of CD44 clustering and its regulation by HA, the reorganization of the cytoskeleton and associated changes in focal adhesion complexes were visualized. Our immunocytochemistry assay revealed that oHA strongly increased intense F-actin stress fibers arranged in spike-like protrusions and the levels of vinculin when cell adhesion were enhanced, whereas nHA showed no effects. Previous reports demonstrated that vinculin is important for the linkage of focal adhesions to the cytoskeleton (57), and vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling (57). The present study indicated that oHA stimulates the assembly of focal adhesion complexes. The effects of oHA treatment on cell adhesion might reflect this reorganization of focal adhesion. Thus, the up-regulation of vinculin expression may be one of the mechanisms by which oHA acts as a promotor of cell adhesion. Taken together, it is likely that the pericellular proteoglycans HA is involved in regulating cell adhesion via their influence on the formation of focal adhesions through their associations with CD44 clustering and cytoskeletal proteins. In fact, it is well known that the response to HA signaling leads to changes in cell motility and adhesion (58). In addition, CD44 interacts directly with ERK1/2 and enhances ERK phosphorylation and cell locomotion (42, 43). To further understand the functional significance of CD44 clustering regulated by nHA or oHA and the mechanism downstream by which these effects are mediated, we examined the ERK1/2 signaling in HK-2 and BT-549 cells. Our findings indicated that oHA induced the activation of ERK, and anti-CD44 mAb blocked the signaling. These results strongly indicate that the stimulating effect of oHA on ERK signaling is mediated through the oHA-CD44 pathway. The utilization of the specific ERK inhibitor resulted in a strong down-regulation of cell adhesive ability, identifying ERK1/2 as a HA-CD44 downstream effector. Our results are consistent with previous report that anti-CD44 antibody or ERK inhibitor inhibited cell adhesion (37, 59), suggesting that pERK1/2, originating from CD44/HA interactions, is an important signal regulating cell adhesion. Based on our results shown above, we propose that nHA interacts with cell surface molecules including CD44 to serve as a “net chain” to link and/or stabilize the existing active molecules such as receptors or transporters on cell surface. As HA and CD44 have been demonstrated to be associated with dynamic movement of cell membrane caveolae or lipid raft within the cell (60–63), molecular size switching on HA may regulate cell metabolism and signaling cooperating with other systems. Further investigation into the role of broken HA in the regulation of cell membrane receptors and transporters is warranted. In summary, our study is the first report to characterize CD44 binding with different size of HA in vivo. We demonstrate that nHA could stimulate cell surface CD44 clustering, which could be further attenuated by oHA. The attenuation could be caused by replacing and competition with the same binding sites of nHA on CD44. We have observed the distribution of the CD44 cluster in human tumor cell line BT549 and human renal proximal tubule cells HK-2, but its relation with tumor progression and inflammation processes needs to be investigated in future. Our novel findings provide more detailed evidence for the interaction between CD44 and nHA or oHA with their influences on cell adhesion and signaling. Supplementary Material Supplemental Data
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Pancreatic stellate cells radioprotect pancreatic cancer cells through β1-integrin signaling.
Tine S Mantoni, Osama Al-Assar, Atsushi Masamune … (2011)
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a strong desmoplastic reaction where the stromal compartment often accounts for more than half of the tumor volume. Pancreatic stellate cells (PSC) are a central mediator of desmoplasia. There is increasing evidence that desmoplasia is contributing to the poor therapeutic response of PDAC. We show that PSCs promote radioprotection and stimulate proliferation in pancreatic cancer cells (PCC) in direct coculture. Our in vivo studies show PSC-dependent radioprotection in response to a single dose and to fractionated radiation. Abrogating β1-integrin signaling abolishes the PSC-mediated radioprotection in PCCs. Furthermore, this effect is independent of PI3K (phosphoinositide 3-kinase) but dependent on FAK. Taken together, we show for the first time that PSCs promote radioprotection of PCCs in a β1-integrin-dependent manner. ©2011 AACR
Author and article information
Journal
Journal ID (nlm-ta): Braz J Med Biol Res
Journal ID (iso-abbrev): Braz. J. Med. Biol. Res
Title:
Brazilian Journal of Medical and Biological Research
Publisher:
Associação Brasileira de Divulgação Científica
ISSN
(Print):
0100-879X
ISSN
(Electronic):
1414-431X
Publication date
(Electronic):
08
May
2015
Publication date Collection:
June
2015
Volume: 48
Issue: 6
Pages: 557-567
Affiliations
[1
]Departamento de Patologia, Faculdade de Medicina, Universidade de São
Paulo, São Paulo, SP, Brasil
[2
]Disciplina de Biologia Molecular, Departamento de Bioquímica,
Faculdade de Medicina, Universidade Federal de São Paulo, São Paulo, SP,
Brasil
[3
]Divisão de Pneumologia, Instituto do Coração, Faculdade de Medicina,
Universidade de São Paulo, São Paulo, SP, Brasil
[4
]Disciplina de Endocrinologia e Metabolismo, Laboratório de
Endocrinologia Molecular e Translacional-LEMT, Departamento de Medicina, Escola
Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP,
Brasil
[6
]Life and Health Sciences Research Institute (ICVS), School of Health
Sciences, University of Minho, Braga, Portugal
Author notes
Correspondence: V.L. Capelozzi, E-mail:
<vcapelozzi@
123456lim05.fm.usp.br>.
Article
DOI: 10.1590/1414-431X20144300
PMC ID: 4470316
PubMed ID: 25992645
SO-VID: 7f96f635-d907-49c8-9836-1bfc306f5bee
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 11
August
2014
Date
accepted
: 17
November
2014
Page count
Figures: 3,
Tables: 3,
Equations: 0,
References: 40,
Pages: 11
Categories
Subject:
Clinical Investigation
Data availability: