- Open Access
Increased FKBP51 in induced sputum cells of chronic obstructive pulmonary disease patients after therapy
European Journal of Medical Research volume 14, Article number: 108 (2009)
Immunophilin FKBP51 assists polypeptide folding, participates in glucocorticoid actions and may play a role in glucocorticoid resistance. FKBP51 is altered in patients with asthma, but its role in chronic obstructive pulmonary disease (COPD) characterized by dysregulation of several pro/antiinflammatory genes is less clear.
We assessed changes in nuclear/cytosolic FKBP51 protein using SDS-PAGE/WB and FKBP51 mRNA by qRT-PCR in cells isolated from induced sputum of stable COPD patients treated with formoterol/budesonide or formoterol/budesonide/theophylline for 4 wk.
Expression of FKBP51 was higher in formoterol/budesonide/theophylline-treated patients, compared with formoterol/budesonide group in both cytosolic and nuclear fractions by about 57% and 31%, respectively (P < 0.001, P < 0.01). FKBP51 mRNA was only slightly, but not significantly, higher in patients on formoterol/budesonide/theophylline.
Increased FKBP51 in COPD patients treated with formoterol/budesonide/theophylline may be important in altering signaling from corticosteroid receptors.
Glucocorticoids effectively switch off pro-inflammatory genes in asthma, but are ineffective in chronic obstructive pulmonary disease (COPD) . It is possible that glucocorticoid resistance in COPD is related to altered glucocorticoid signaling. FK506-binding protein (FKBP), the peptidyl prolyl cis-trans isomerase and the member of a large immunophilin family assists proper folding of polypeptides, responds to bronchodilator drugs and alters glucocorticoid effects [2, 3]. There are two functional FKBP proteins interacting with glucocorticoid receptor (GR)-Hsp90 complex: FKBP51 coded by FKBP5 gene, which binds unliganded GR and FKBP52 coded by FKBP4 gene, which interacts with liganded GR and activate GR complex . This complex called transportosome is transported to the nucleus and transactivates or transrepresses specific genes or transcription factors . It has been shown that increased levels of FKBP51 caused glucocorticoid resistance in New World primates . The role of FKBP51 in COPD, characterized by disregulation of several pro/anti-inflammatory genes and signaling proteins remain largely unknown, but expression of FKBP51 is altered in asthma  and affected by glucocorticoids [4, 7], theophylline (Th) , or inhaled β2-receptor agonists .
Theophylline may restore steroid responsiveness in COPD patients via normalization of reduced histone deacetylase [1, 10], the drug has both antiinflammatory and antioxidant properties and affects glucocorticoid response [11, 12]. The possible Th-dependent pathway involves increased cyclic AMP, activation of cyclic AMP response element binding protein (CREB) and chaperone system , since the levels of phosphorylated CREB correlated with increased expression of human hsp90β gene . The goal of the present study was to assess FKBP51 protein expression and nuclear/cytosolic protein distribution and possible changes in FKBP51 mRNA in cells isolated from induced sputum of stable COPD patients treated with formoterol/budesonide or formoterol/budesonide/theophylline.
Subjects and methods
All patients included in the study gave their consent after a full discussion of the nature of the study and the study was approved by a local Ethics Committee.
Sputum was induced in 36 COPD patients with stable disease, defined according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines . All patients with COPD had airflow limitation (FEV1 < 80% predicted, FEV1/FVC < 70%, GOLD stage 2-2) and received no COPD therapy for 4 wk. Lung function and DLCO tests were performed with a body box (Elite DL, Medgraphics, USA). The measurement was performed using standard protocols according to American Thoracic Society guidelines. All subjects were characterized with respect to sex, age, smoking history, COPD symptoms, comorbidity, and current medical treatment. Exclusion criteria included the following: other systemic diseases, other lung diseases apart from COPD and lung tumors, pulmonary infection, and antibiotic treatment 4 wk before inclusion or inhaled or oral glucocorticoids in the 3 mo before inclusion. No patient in the study had symptoms nor was treated for COPD exacerbation during at least 2 mo preceding the day of inclusion.
All patients underwent a 4 wk washout salbutamol only on demand therapy. At the beginning of the treatment patients were stratified to following treatments: formoterol/budesonide (F/ICS, n = 18) and formoterol/budesonide/theophylline (F/ICS/Th); n = 18) b.i.d. for 4 wk.
Sputum Induction and Processing
Sputum was induced by the inhalation of a 4.5% hypertonic aerosol saline solution, which was generated by an ultrasonic nebulizer (Voyager, Secura Nova; Warsaw, Poland) . Samples were processed within 15 min after the termination of induction. Throughout the procedure, subjects were encouraged to cough and to expectorate into a plastic container. Three flow volume curves were performed before and after each inhalation, and the best FEV1 was recorded. Induction of sputum was stopped if the FEV1 value fell by at least 20% from baseline or if troublesome symptoms occurred.
Induced sputum samples were processed to isolate mRNA (qPCR-grade RNA isolation kit, SABiosciences, Frederick, USA) or were homogenized for 1 min in the lysis buffer containing 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM NaF, 50 mM -glycerophosphate, a protease inhibitor tablet, 0.2 mM Na orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotenin, and 10% Nonidet P-40. The samples were then incubated on ice for 15 min and centrifuged at 13000 × g for 30 s. The cell pellets containing nuclei were retained and resuspended in extracting buffer (50 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 50 mM KCl, 300 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 0.2 mM NaF, 0.2 mM Na-orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotenin, 50 mM - glycerophosphate, and a protease inhibitor tablet (Complete Mini; Roche Diagnostics, Mannheim, Germany). The samples were then incubated on a rotating platform for 30 min at 4°C followed by centrifugation at 13000 × g for 5 min. The resulting nuclear and cytosolic fractions were evaluated for the expression of FKBP51.
Protein levels were measured using a BCA kit (Sigma-Aldrich, Poznan, Poland).
FKBP51 Protein Levels
FKBP51 protein levels were assessed in nuclear and cytosolic fractions using SDS/PAGE/Western blots. Nuclear or cytosolic proteins were separated by SDS/PAGE in reducing conditions, transferred onto polyvinylidene difluoride membranes, and incubated with specific rabbit antibodies against human FKBP51 (Abcam, Cambridge, USA). After washing, bound antibody was detected using appropriate secondary antirabbit antibody (Abcam) linked to horseradish peroxidase. The bound complexes were detected using enhanced chemiluminescence (ECL, Amersham, GE Healthcare, Little Chalfont, UK) and quantified using Image Quant software. The constitutively expressed protein, β-actin, served as a loading control and the data were quantified in respect to β-actin expression.
Expression of FKBP5 gene coding for immunophilin FKBP51 was assessed using SYBR Green based qRTPCR after total RNA extraction from induced sputum cells, RNA purification and template cDNA synthesis was performed with commercial PCR master mixes containing appropriate controls and reference dyes (SABiosciences, Frederic, USA) as described earlier . Samples were run on ABI 7900HT instrument and data were analyzed and quantified using SABiosciences software based on the ΔΔCt method with normalization of the raw data to either housekeeping genes or an external RNA control.
Data are expressed as means ± SD. Statistical significance was calculated using one-way analysis of variance ANOVA, followed by Bonferroni post-hoc test for selected pairs of data.
Table 1 demonstrates the cytosolic and nuclear FKBP51 proteins and the FKBP51 mRNA in cells of patients treated with F/ICS and F/ICS/Th for 4 wk. Corresponding representative Western Blot pictures are shown in Figure 1. The expression of FKBP51 was higher in the F/ICS/Th-treated patients, compared with the F/ICS treated patients in both cytosolic and nuclear fractions by about 58% (P < 0.01) and 31% (P < 0.05), respectively. The cytosolic/nuclear FKBP51 ratio was significantly (P < 0.01) increased in the F/ICS/Th patients compared with the F/ICS-treated patients indicating relative accumulation of FKBP51 in the cytosol. FKBP51 mRNA in cells isolated from the F/ICS/Th patients was highly variable between patients, and although the mean expression in this group was slightly higher than in F/ICS patients, the difference was not statistically significant.
Theophylline, a non-specific phosphodiesterase inhibitor, is a narrow therapeutic index drug and must be monitored due to significant side effects. In COPD, theophylline is used as one of main bronchodilators, aside from inhaled anticholinergics and long acting β2 agonists. It has been shown that thophylline withdrawal in severe COPD patients may worsen their clinical status [18, 19]. Thephylline signaling pathways are not clearly identified, but experimental and clinical data indicate that it has a broad spectrum of actions including antioxidant, immunomodulatory, and antiinflammatory effects [11, 12]. It inhibits activation of nuclear transcription factor NF-κB in human pulmonary epithelial cell  and may even reverse steroid resistance in COPD . It has been shown, that theophylline alters the expression of several genes, downregulates IL-13 mRNA, and upregulates mRNA of FKBP38-cochaperone protein involved in apoptosis . In the present study, the expression of cochaperone immunophilin FKBP51 was higher in F/ICS/Th-treated patients, compared with the F/ICS group in both cytosolic and nuclear fractions, but increased cytosolic/nuclear FKBP51 ratio indicates that FKBP51 is mostly accumulated in the cytosol. It is possible that this increase may be transcriptionally regulated, which, however, should be verified on higher numbers of patients. GR-GC-heat shock protein 90kD (Hsp90) complex-associated immunophilins compete with each other and it is possible that the relative concentrations of the different immunophilins bound to the complex might control GR activation. Thus, the possible functional consequence of increased FKBP51 protein may be an altered nuclear transport of GR . FKBP51 is a major immunophilin in inactive GR-Hsp90 complexes, and in the presence of glucocorticoids it is replaced by another cochaperone - FKBP52 - with subsequent nuclear translocation [4, 21]. Mouse knockout experiments support a major role of FKBP52 in GR activation [22, 23], while other studies point to the contribution of elevated FKBP51 protein to glucocorticoid resistance . The present results indicate that high doses of theophylline in combined therapy with b2agonists and glucocorticoids may possibly decrease the glucocorticoid response via discriminative FKBP51 binding, but this is not consistent with the published data indicating that theophylline can improve clinical status of some COPD patients or restore the antiinflammatory action of glucocorticoids in cells subjected to oxidative stress [1, 10]. Interestingly, steroid hormones themselves regulate immunophilin levels and may increase systemic FKBP51 expression, producing partial functional feedback inhibition of GR [24, 25]. Thus, the present study indicates that theophylline and glucorticoids in polytherapy may act synergistically in this aspect, but functional consequences of increased FKBP51 in COPD patients remain to be established.
Conflicts of interest
The authors declare that they have no competing interests.
Barnes PJ, Adcock IM: Glucocorticoid resistance in inflammatory diseases. Lancet 2009, 373: 1905–17. Review 10.1016/S0140-6736(09)60326-3
Schiene-Fischer C, Yu C: Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett 2001, 495: 1–6. Review 10.1016/S0014-5793(01)02326-2
Kang CB, Hong Y, Dhe-Paganon S, Yoon HS: FKBP family proteins: immunophilins with versatile biological functions. Neurosignals 2008, 16: 318–25. Review 10.1159/000123041
Zhang X, Clark AF, Yorio T: FK506-binding protein 51 regulates nuclear transport of the glucocorticoid receptor beta and glucocorticoid responsiveness. Invest Ophthalmol Vis Sci 2008, 49: 1037–47. 10.1167/iovs.07-1279
Woodruff PG, Boushey HA, Dolganov GM, Barker CS, Yang YH, Donnelly S, Ellwanger A, Sidhu SS, Dao-Pick TP, Pantoja C, Erle DJ, Yamamoto KR, Fahy JV: Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci USA 2007, 104: 15858–63. 10.1073/pnas.0707413104
Davies TH, Ning YM, Sánchez ER: Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506. Biochemistry 2005, 44: 2030–8. 10.1021/bi048503v
Bolger GB, Peden AH, Steele MR, MacKenzie C, McEwan DG, Wallace DA, Huston E, Baillie GS, Houslay MD: Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2. Biol Chem 2003, 278: 33351–63. 10.1074/jbc.M303269200
Kaur M, Chivers JE, Giembycz MA, Newton R: Long-acting beta2-adrenoceptor agonists synergistically enhance glucocorticoid-dependent transcription in human airway epithelial and smooth muscle cells. Mol Pharmacol 2008, 73: 203–14.
Scammell JG, Denny WB, Valentine DL, Smith DF: Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates. Gen Comp Endocrinol 2001, 124: 152–65. 10.1006/gcen.2001.7696
Marwick JA, Wallis G, Meja K, Kuster B, Bouwmeester T, Chakravarty P, Fletcher D, Whittaker PA, Barnes PJ, Ito K, Adcock IM, Kirkham PA: Oxidative stress modulates theophylline effects on steroid responsiveness. Biochem Biophys Res Commun 2008, 377: 797–802. 10.1016/j.bbrc.2008.10.065
Mroz RM, Holownia A, Chyczewska E, Braszko JJ: Chronic obstructive pulmonary disease: an update on nuclear signaling related to inflammation and anti-inflammatory treatment. J Physiol Pharmacol 2008,59(Suppl 6):35–42. Review
Cosio BG, Iglesias A, Rios A, Noguera A, Sala E, Ito K, Barnes PJ, Agusti A: Low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of COPD. Thorax 2009, 64: 424–9. 10.1136/thx.2008.103432
Mroz RM, Holownia A, Chyczewska E, Drost EM, Braszko JJ, Noparlik J, Donaldson K, Macnee W: Cytoplasm-nuclear trafficking of CREB and CREB phosphorylation at Ser133 during therapy of chronic obstructive pulmonary disease. J Physiol Pharmacol 2007, 58: 437–44.
Bingsheng L, Ninghua W, Yufei S: Cyclic AMP response element binding protein (CREB) participates in the heat inducible expression of human hsp90 gene Chinese Science Bulletin. 2001, 46: 1645–8.
From the Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2008 [http://www.goldcopd.org]
Loh LC, Kanabar V, D'Amato M, Barnes NC, O'Connor BJ: Sputum induction in corticosteroid-dependant asthmatics: risks and airway cellular profile. Asian Pac J Allergy Immunol 2005, 23: 189–96.
Holownia A, Mroz RM, Noparlik J, Chyczewska E, Braszko JJ: Expression of CREB-binding protein and peroxisome proliferator-activated receptor gamma during formoterol or formoterol and corticosteroid therapy of chronic obstructive pulmonary disease. J Physiol Pharmacol 2008,59(Suppl 6):303–9.
Caramori G, Adcock I: Pharmacology of airway inflammation in asthma and COPD. Pulm Pharmacol Ther 2003, 16: 247–277. Review 10.1016/S1094-5539(03)00070-1
Barnes PJ: Theophylline for COPD. Thorax 2006, 61: 742–3. Review 10.1136/thx.2006.061002
Ichiyama T, Hasegawa S, Matsubara T, Hayashi T, Furukawa S: Theophylline inhibits NF-kappa B activation and I kappa B alpha degradation in human pulmonary epithelial cells. Naunyn Schmiedebergs Arch Pharmacol 2001, 364: 558–61. 10.1007/s00210-001-0494-x
Yao PL, Tsai MF, Lin YC, Wang CH, Liao WY, Chen JJ, Yang PC: Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation. Respir Res 2005, 6: 89. 10.1186/1465-9921-6-89
Wolf IM, Periyasamy S, Hinds T Jr, Yong W, Shou W, Sanchez ER: Targeted ablation reveals a novel role of FKBP52 in gene-specific regulation of glucocorticoid receptor transcriptional activity. J Steroid Biochem Mol Biol 2009, 113: 36–45. 10.1016/j.jsbmb.2008.11.006
Yong W, Yang Z, Periyasamy S, Chen H, Yucel S, Li W, Lin LY, Wolf IM, Cohn MJ, Baskin LS, Sánchez ER, Shou W: Essential role for Co-chaperone Fkbp52 but not Fkbp51 in androgen receptor-mediated signaling and physiology. J Biol Chem 2007, 282: 5026–36.
Zhu W, Zhang JS, Young CY: Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP Carcinogenesis. 2001, 22: 1399–403.
Vermeer H, Hendriks-Stegeman BI, van der Burg B, van Buul-Offers SC, Jansen M: Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glucocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab 2003, 88: 277–284. Cell Death Differ 2004; 11: 45–55 10.1210/jc.2002-020354
Supported by grant N402 074 32/2410 from Ministery of Science and Education in Poland.
About this article
Cite this article
Holownia, A., Mroz, R.M., Kolodziejczyk, A. et al. Increased FKBP51 in induced sputum cells of chronic obstructive pulmonary disease patients after therapy. Eur J Med Res 14, 108 (2009) doi:10.1186/2047-783X-14-S4-108