FK506-binding Protein 51 mRNA Research Articles

Bta-miR-365-3p-targeted FKBP5 participates in the AMPK/mTOR signaling pathway in the regulation of preadipocyte differentiation in cattle.

MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. We identified bta-miR-365-3p, which specifically targets the 3'untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5),and verified its mechanisms for regulating expression and involvement in adipogenesis. In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-365-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dual-luciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by qRT-PCR and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. AMPK/mTOR signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.

FKBP51 Contributes to Uterine Leiomyoma Pathogenesis by Inducing Cell Proliferation and Extracellular Matrix Deposition

The FK506-binding protein 51 (FKBP51) binds progesterone receptor (PR), glucocorticoid receptor (GR), and androgen receptor (AR) to coregulate their transcriptional activity. We evaluated FKBP51 expression and function in human leiomyoma vs. myometrial tissues and primary cultures to discover FKBP51 role(s) in the pathogenesis of leiomyomas. Quantification of in situ FKBP51 mRNA and protein levels inpaired myometrial vs. leiomyoma tissues from proliferative and secretory phases were analyzed by qPCR (n = 14), immunoblotting (n = 20), and immunohistochemistry (n = 12). Control (scramble) vs. FKBP5 siRNA-transfected leiomyoma cell cultures were assessed for proliferation, apoptosis, and mRNA levels of genes involved in cell survival and extracellular matrix (ECM) formation. Significantly higher FKBP5 mRNA levels were detected in leiomyoma vs. paired myometrium (P < 0.001). Immunoblot (P = 0.001) and immunostaining (P ≤ 0.001) confirmed increased FKBP51 levels in leiomyoma vs. paired myometrium. Compared to control siRNA transfection, FKBP5-silenced leiomyoma cell cultures displayed significantly decreased cell survival factors and reduced proliferation (P < 0.05). Moreover, qPCR analysis revealed significantly lower mRNA levels of ECM, TIPM1, and TIPM3 proteins in FKBP5-silenced leiomyoma cell cultures (P < 0.05). Increased FKBP51 expression in leiomyoma likely involves dysregulation of steroid signaling by blocking GR and PR action and promoting proliferation and ECM production. Evaluating the effect of FKBP51 inhibition in preclinical studies will clarify its significance as a potential therapeutic approach against leiomyoma.Supplementary InformationThe online version contains supplementary material available at 10.1007/s43032-022-00921-2.

Effect of dihydrotestosterone fluctuation on mismatch repair gene mutL homolog 1 in prostate epithelial cells and its mechanism

Objective To investigate the relationship between the fluctuation of dihydrotestosterone concentration and the expression of mutL homolog 1 (MLH1) in mismatch repair system in prostate epithelial cells (RWPE-2 cell line) and its mechanism. Methods RWPE-2 cells were randomly divided into blank group [anhydrous ethanol solvent and dihydrotestosterone (DHT) concentration of 0 nmol/L], constant concentration group (concentration of DHT: 1, 10 nmol/L) and fluctuating concentration group (concentration of DHT: 1, 10 nmol/L, fluctuating every 24 h). After different time (0, 24, 48 and 72 h), cell counting kit-8 was use to examine cell proliferation and the target genes in androgen receptor (AR) signaling pathway [prostate specific antigen (PSA), FK506 binding protein 51 (FKBP5)], protein kinase B (Akt) signaling pathway [Cyclin D1, B cell lymphoma/leukemia-2 (bcl-2)] and MLH1 was detected by Western blotting, real-time quantitative polymerase chain reaction (Real-time PCR) and immunofluorescence. Graph Pad 7.0 statistical software was used for analysis and differences between groups were analyzed by t test. Results The morphological changes of RWPE-2 cells had a certain concentration-dependent relationship with DHT: constant group and fluctuating group had more cells and better growth than blank group. After RWPE-2 cells were stimulated with DHT for 72 h, as compared with the blank group (6.904±0.143), the relative A values of the constant group [(7.073±0.103) and (8.508±0.187)] and the fluctuation group [(8.662±0.327) and (9.239±0.167)] increased statistically (t=6.805, 4.922, 10.6, P<0.01). Western blotting and RT-qPCR results confirmed that the mRNA and protein levels of every target gene were increased significantly in constant group and fluctuating group (P<0.05) as compared with blank group. Meanwhile, the difference in fluctuating group was more significant than constant group. Whereas, FKBP5 mRNA decreased in fluctuating group as compared with constant group (t=7.101, 10.760, 8.289, 8.088, 8.519, 9.157, 11.330, P<0.05). The distribution of above genes increased in the nucleus after DHT intervention with more significant difference in fluctuating group. Conclusion DHT can up-regulate the expression of the mismatch repair protein MLH1 in RWPE-2 cells, meanwhile, DHT fluctuations can increase this trend and AR and Akt signaling pathways-mediated proliferation is one of the mechanisms. Key words: Dihydrotestosterone fluctuation; Cell proliferation; Mismatch repair; Androgen receptor signal pathway; Protein kinase B signal pathway

Adverse early life environment induces anxiety-like behavior and increases expression of FKBP5 mRNA splice variants in mouse brain.

Adverse early life environment (AELE) predisposes adult offspring toward anxiety disorders. Anxiety disorders are associated with prenatal injuries in key regions of the brain including prefrontal cortex (PFC), hippocampus (HP), and hypothalamus (HT). Injuries in these brain regions result in an impaired hypothalamus-pituitary-adrenal axis (HPA axis) and stress response. An important regulator of the stress response is FK506-binding protein 5 (FKBP5). FKBP5 is a cochaperone of the glucocorticoid receptor (GR) and inhibits GR-mediated regulatory feed-back on the HPA axis in response to stress. Human studies have shown that polymorphisms of FKBP5 are associated with higher FKBP5 levels. Increased FKBP5 leads to GR resistance and impaired negative feedback, which is associated with anxiety disorders. FKBP5 and its mRNA splice variants in the aforementioned brain regions have not been reported. We hypothesized that AELE will increase expression of FKBP5 and its mRNA splice variants in PFC, HP, and HT as well as increase anxiety in adult mice. AELE increased expression of FKBP5 and its mRNA variants in PFC, HP and HT at postnatal day 21. Additionally, AELE caused anxiety and increased GR abundance in association with these changes in FKBP5 expression. We speculate that these changes in FKBP5 mRNA variants affect HPA axis function and contributes to subsequent anxiety-like behavior later in life in AELE mice.

P-172 - Modulation of FKBP5 Expression by cigarette smoke

Cigarette smoke (CS) is one of the most toxic inhaled environmental stressors leading to numerous, preventable, human pathologies. Its deleterious effects on neuro-immunomodulatory systems remain to be documented. We hypothesize that CS activates hypothalamus-pituitary-adrenal axis (HPA axis) releasing glucocorticoids by adrenal cortex. FK506 binding protein 5 (FKBP5), also known as FKBP51 (family of immunophilins) are transcriptionally induced by glucocorticoids and is known to regulate glucocorticoid receptor (GR) responses. Increased expression of FKBP5 was reported to confer ‘glucocorticoid resistance’ by binding to GR and preventing GR-translocation to nucleus. Our focus on FKBP5 was stimulated by data from a genome-wide search for mRNA correlates of (CS)-responsive lung transcriptomes. Studies comparing the lung transcriptomic profile of CS-exposed C57BL6 and AJ mice to air-breathing controls showed elevated levels of FKBP5 mRNA expression in both mouse strains. FKBP5 was also seen to regulate IκBα and p65 nuclear translocation in lung cancer cell lines, suggesting a contributory role of FKBP5 in proinflammatory stimuli of CS. The data suggests that modulation of FKBP5 expression by CS may cause dysregulated GR responses and modulate inflammatory-immune pathways in lungs.

Hyperforin and Miquelianin from St. Johnʼs Wort Attenuate Gene Expression in Neuronal Cells After Dexamethasone-Induced Stress

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis plays an important part in the development of depressive symptoms. In this study, the effects of a commercial St.John's wort extract (STW3-VI), hyperforin, miquelianin, and the selective serotonin reuptake inhibitor citalopram on the expression of genes relevant to HPA axis function were investigated in human neuronal cells. SH-SY5Y cells were treated with STW3-VI (20 µg/mL), hyperforin (1 µM), miquelianin (10 µM), or citalopram (10 µM) in the presence of the glucocorticoid receptor agonist dexamethasone (DEX,10 µM) for 6 h and 48 h, respectively. Quantitative real-time polymerase chain reaction was used to determine the expression of FKBP5 (FK506 binding protein 51), CREB (cAMP responsive element binding protein), GRIK4 (glutamate ionotropic receptor kainate type subunit 4), VEGF (vascular endothelial growth factor), NET (norepinephrine transporter), and ARRB (β-arrestins), promising biomarkers of antidepressant therapy. Using DEX to mimic stress conditions, it was shown that the gene expression pattern of FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 in SH-SY5Y cells is time- and treatment-dependent. Most pronounced effects were observed for FKBP5: after 6 h of co-incubation, only STW3-VI could reverse the DEX-induced increase in FKBP5 expression, and after 48 h, citalopram, miquelianin, and hyperforin also reversed the glucocorticoid-induced increase in FKBP5 mRNA expression. The effects observed on FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 are in good correlation with published data, suggesting that this in vitro model could be used to screen the responsiveness of antidepressants under stress conditions.

FKBP51 Immunohistochemical Expression: A New Prognostic Biomarker for OSCC?

Up-to-date, several molecular markers of prognosis have been studied in Oral Squamous Cell Carcinoma (OSCC), but none entered in the clinical setting. Therapy of OSCC tumors mainly relies on surgery, radiotherapy and partially on chemotherapy; there is an urgent need for biomarkers able to better stratify OSCC patients’ risk to address targeted therapeutic strategies. The role of immune response in the pathogenesis and biological behavior of OSCC has been investigated by several authors, and promising results have been obtained with immune checkpoint inhibitors. We already investigated the role of the immune modulator FK506-binding protein 51 (FKBP51), a FK506-binding immunophilin, in cutaneous melanoma biology, and its expression in several human solid tumors. In the present study, we aimed to assess the value of FKBP51 expression in OSCC tumor cells as a marker of outcome. We collected clinical data from 72 patients who underwent surgery for Squamous Cell Carcinoma (SCC) of the tongue, floor, lips and palate. FKBP51 expression was assessed by immunohistochemistry on paraffin-embedded tumor tissues. In addition, we evaluated the human papillomavirus (HPV) status of primary tumors by immunohistochemistry, viral subtyping and In Situ Hybridization (ISH) assay. We found that high FKBP51-expressing tumors characterized the OSCCs with the worst prognosis: the high immunohistochemical expression of FKBP51 associated with death occurring within five years from the diagnosis with a sensitivity of 88.46% and a specificity of 91.67%. The estimated positive predictive value of the test was 88.45% and negative predictive value 91.67%. We tested FKBP51 mRNA presence, by RT-PCR assay, in a selected series of OSCC tumors, and we found that mRNA correlated well to the protein expression and to the clinical outcome. Applying the Bayes formula, we estimated an 88% probability of dying within five years from the diagnosis of OSCC patients with a high FKBP51 immunohistochemical (IHC) test result (>51% of FKBP51 positive tumor cells). On the basis of our analysis, we propose tumor tissue expression of FKBP51 protein as a reliable prognostic marker for OSCC tumors.

Abstract # 1845 Age- and depression-related changes in FKBP4 and FKBP5 expression across adolescence

FK506-binding protein 5 (FKBP5) has repeatedly been shown to be a critical determinant of depression. For example, FKBP5 polymorphisms emerged as moderators of depression risk and protein levels have been linked to disease severity. However, little is known about FKBP5 in the context of depression pathogenesis. To address this gap, we repeatedly assessed 42 female adolescents (14-19yrs) at high risk for depression over the course of 2.5 years. Every 6 months, participants underwent a diagnostic interview and venipuncture to measure FKBP5 and FKBP4 mRNA levels. To assess the role of age, four equally-sized age groups were formed (14–15, 16–17, 18, 19 yrs). Over the study period, 21 adolescents showed at least one episode of depression (DEP), while 21 stayed symptom-free (SF). FKBP5 expression increased across age groups independent of health status, such that 14–15yrs old DEP and SF adolescents showed the lowest and 19yrs old DEP and SF adolescents showed the highest FKPB5 expression across time (age: F = 3.22, p = .035). While a similar pattern emerged for FKBP4 in SF adolescents, DEP adolescents exhibited overall lower as well as less age-variable FKBP4 mRNA levels (FBKP4-by-age-by-health status: F = 2.28, p = .023). In summary, while changes in FKBP4 and FKBP5 expression across adolescence showed a similar up-regulatory pattern in healthy participants, depression was associated with a potential de-synchronization of the two co-chaperons suggesting reduced glucocorticoid receptor signaling.

FKBP5 genotype-dependent DNA methylation and mRNA regulation after psychosocial stress in remitted depression and healthy controls.

Background:Polymorphisms in the FK506 binding protein 5 (FKBP5) gene have been shown to influence glucocorticoid receptor sensitivity, stress response regulation, and depression risk in traumatized subjects, with most consistent findings reported for the functional variant rs1360780. In the present study, we investigated whether the FKBP5 polymorphism rs1360780 and lifetime history of major depression are associated with DNA methylation and FKBP5 gene expression after psychosocial stress.Methods:A total of 116 individuals with a positive (n = 61) and negative (n = 55) lifetime history of major depression participated in the Trier Social Stress Test. We assessed plasma cortisol concentrations, FKBP5 mRNA expression, and CpG methylation of FKBP5 intron 7 in peripheral blood cells.Results:Genotype-dependent plasma cortisol response to psychosocial stress exposure was observed in healthy controls, with the highest and longest-lasting cortisol increase in subjects with the TT genotype of the FKBP5 polymorphism rs1360780, and healthy controls carrying the T risk allele responded with a blunted FKBP5 mRNA expression after psychosocial stress. No genotype effects could be found in remitted depression.Conclusions:The FKBP5 rs1360780 polymorphism is associated with plasma cortisol and FKBP5 mRNA expression after psychosocial stress in healthy controls but not in remitted depression. Preliminary results of the DNA methylation analysis suggest that epigenetic modifications could be involved.

Early life stress and serotonin transporter gene variation interact to affect the transcription of the glucocorticoid and mineralocorticoid receptors, and the co-chaperone FKBP5, in the adult rat brain.

The short allelic variant of the serotonin transporter (5-HTT) promoter-linked polymorphic region (5-HTTLPR) has been associated with the etiology of major depression by interaction with early life stress (ELS). A frequently observed endophenotype in depression is the abnormal regulation of levels of stress hormones such as glucocorticoids. It is hypothesized that altered central glucocorticoid influence on stress-related behavior and memory processes could underlie the depressogenic interaction of 5-HTTLPR and ELS. One possible mechanism could be the altered expression of the genes encoding the glucocorticoid and mineralocorticoid receptors (GR, MR) and their inhibitory regulator FK506-binding protein 51 (FKBP5) in stress-related forebrain areas. To test this notion, we exposed heterozygous (5-HTT+/−) and homozygous (5-HTT−/−) serotonin transporter knockout rats and their wildtype littermates (5-HTT+/+) to daily 3 h maternal separations from postnatal day 2 to 14. In the medial prefrontal cortex (mPFC) and hippocampus of the adult male offspring, we found that GR, MR, and FKBP5 mRNA levels were affected by ELS × 5-HTT genotype interaction. Specifically, 5-HTT+/+ rats exposed to ELS showed decreased GR and FKBP5 mRNA in the dorsal and ventral mPFC, respectively. In contrast, 5-HTT+/− rats showed increased MR mRNA levels in the hippocampus and 5-HTT−/− rats showed increased FKBP5 mRNA in the ventral mPFC after ELS exposure. These findings indicate that 5-HTT genotype determines the specific adaptation of GR, MR, and FKBP5 expression in response to early life adversity. Therefore, altered extra-hypothalamic glucocorticoid signaling should be considered to play a role in the depressogenic interaction of ELS and 5-HTTLPR.

Effect of the common functional FKBP5 variant (rs1360780) on the hypothalamic-pituitary-adrenal axis and peripheral blood gene expression

Regulation of hypothalamic-pituitary-adrenal (HPA) axis reactivity plays an important role in the development of stress-related psychiatric disorders. FK506 binding protein 5 (FKBP5) modulates HPA axis reactivity via glucocorticoid receptor (GR; NR3C1) sensitivity and signaling. The T allele of the single nucleotide polymorphism, FKBP5 rs1360780 (C/T), is associated with higher FKBP5 induction by glucocorticoids. In the present study, we performed the dexamethasone/corticotropin releasing hormone (DEX/CRH) test and quantitative real-time PCR analysis of peripheral blood mononuclear cell (PBMC) cDNA samples in 174 and 278 non-clinical individuals, respectively. We found increased suppression of the stress hormone (cortisol) response to the DEX/CRH test (P=0.0016) in aged (>50 years) individuals carrying the T allele compared with aged non-T allele carriers. T carriers showed significant age-related changes in GR and FKBP5 mRNA expression levels in PBMCs (P=0.0013 and P=0.00048, respectively). Our results indicate that FKBP5 rs1360780 regulates HPA axis reactivity and expression levels of GR and FKBP5 in PBMCs in an age-dependent manner. Because these phenotypes of aged T carriers are similar to endophenotypes of people with post-traumatic stress disorder, our findings may be useful for determining the molecular mechanisms, treatment, and preventive strategies for this disease.

Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation

Objectives We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34+ cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34+ cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation.Methods Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-κB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay.Results FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl l-cysteine. Finally, glucocorticoid enhanced FKBP5 mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner.Conclusions These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-κB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis.

Association of Eosinophilic Inflammation with FKBP51 Expression in Sputum Cells in Asthma

BackgroundAirway eosinophilia is a predictor of steroid responsiveness in steroid-naïve asthma. However, the relationship between airway eosinophilia and the expression of FK506-binding protein 51 (FKBP51), a glucocorticoid receptor co-chaperone that plays a role in steroid insensitivity in asthma, remains unknown.ObjectiveTo evaluate the relationship between eosinophilic inflammation and FKBP51 expression in sputum cells in asthma.MethodsThe FKBP51 mRNA levels in sputum cells from steroid-naïve patients with asthma (n = 31) and stable asthmatic patients on inhaled corticosteroid (ICS) (n = 28) were cross-sectionally examined using real-time PCR. Associations between FKBP51 levels and clinical indices were analyzed.ResultsIn steroid-naïve patients, the FKBP51 levels were negatively correlated with eosinophil proportions in blood (r = −0.52) and sputum (r = −0.57), and exhaled nitric oxide levels (r = −0.42) (all p<0.05). No such associations were observed in patients on ICS. In steroid-naïve patients, improvement in forced expiratory volume in one second after ICS initiation was correlated with baseline eosinophil proportions in blood (r = 0.74) and sputum (r = 0.76) and negatively correlated with FKBP51 levels (r = −0.73) (all p<0.0001) (n = 20). Lastly, the FKBP51 levels were the lowest in steroid-naïve asthmatic patients, followed by mild to moderate persistent asthmatic patients on ICS, and the highest in severe persistent asthmatic patients on ICS (p<0.0001).ConclusionsLower FKBP51 expression in sputum cells may reflect eosinophilic inflammation and glucocorticoid responsiveness in steroid-naïve asthmatic patients.

Abstract 3559: FKBP5 is an androgen-responsive gene in thyroid cancer cells.

Abstract There has been a steady increase in the incidence thyroid cancer in the United States, with over 56,000 cases predicted for 2012. The increased aggressiveness of the disease and poorer clinical outcome in men and postmenopausal women may be due to androgen/androgen receptor (AR) related functions. FK506 binding protein 51 (FKBP5) is a member of the immunophilin family and plays a role in steroid receptor function. Further, FKBP5 regulates the NFkB and AKT pathways and is thought to be relevant to the neoplastic process. Immunohistochemical analysis of 38 papillary thyroid cancer (PTC) specimens indicated that androgen receptor is expressed in both normal and cancerous epithelial cells, in both men and women. Analysis of AR expression in thyroid cell lines indicated that the normal thyroid cell line, Nthy-ori 3-1 and PTC cell lines BCPAP and TPC-1 expressed very low levels of AR, as measured by qRT-PCR and western blotting (&amp;gt;1000 fold less than the prostate cancer cell line, LNCaP). In contrast, PTC/anaplastic 8505C cells expressed higher levels of AR protein (approximately one third the level expressed in LNCaP cells). Upon addition of androgen, AR in 8505C translocated into the nucleus (as determined by immunocytochemistry) and upregulated transcription of both AR (∼1.7 fold) and FKBP5 (∼1.8 fold). To more fully understand the function of androgen/AR in thyroid cells, the AR cDNA from pSG5-AR was subcloned into the pcDNA3.1+ expression vector and stably transfected into thyroid cell lines. Primers for qRT-PCR of the endogenous or the transfected gene were designed. The 84E7 clone of transfected 8505C cells expressed 68 fold higher levels of AR mRNA than 8505C and 16 fold higher AR protein expression (∼5 fold higher AR protein expression than LNCaP). Upon addition of DHT to clone 84E7, AR translocated into the nucleus, and increased AR and FKBP5 mRNA expression (∼3 fold and ∼13 fold, respectively). The association between androgens/AR and upregulation of FKBP5 in thyroid cancer cells suggests that FKBP5 may play a role in thyroid cancer etiology and the different clinical presentation and outcomes between young women and men/postmenopausal women. Citation Format: Melanie E. MacEwan, Ken Wong, Ismatun Swati, Suqing Xie, Mohammad Rasul, Olena Ardacheva, Joseph Buchsbaum, Edward Shin, Augustine Moscatello, Stimson Schantz, Raj K. Tiwari, Jan Geliebter. FKBP5 is an androgen-responsive gene in thyroid cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2013-3559

Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation

We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34(+) cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34(+) cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation. Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-κB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay. FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl L-cysteine. Finally, glucocorticoid enhanced FKBP5 mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner. These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-κB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis.

Differences in FKBP51 Regulation Following Chronic Social Defeat Stress Correlate with Individual Stress Sensitivity: Influence of Paroxetine Treatment

Various clinical studies have identified FK506-binding protein 51 (FKBP51) as a target gene involved in the development of psychiatric disorders such as depression. Furthermore, FKBP51 has been shown to affect glucocorticoid receptor signaling by sensitivity modulation and it is implicated in stress reactivity as well as in molecular mechanisms of stress vulnerability and resilience. We investigated the physiological, behavioral, and neuroendocrine parameters in an established chronic stress model both directly after stress and after a recovery period of 3 weeks and also studied the efficacy of paroxetine in this model. We then examined FKBP51 mRNA levels in the dorsal and ventral part of the hippocampus and correlated the expression to behavioral and endocrine parameters. We show robust chronic stress effects in physiological, behavioral, and neuroendocrine parameters, which were only slightly affected by paroxetine treatment. On the contrary, paroxetine led to a disruption of the neuroendocrine system. FKBP51 expression was significantly increased directly after the stress period and correlated with behavioral and neuroendocrine parameters. Taken together, we were able to further elucidate the role of FKBP51 in the mechanisms of stress resilience and vulnerability, especially with respect to behavioral and neuroendocrine parameters. These findings strongly support the concept of FKBP51 as a marker for glucocorticoid receptor sensitivity and its involvement in the development of psychiatric disorders.

Corticosteroid‐induced gene expression in allergen‐challenged asthmatic subjects taking inhaled budesonide

Inhaled corticosteroids (ICS) are the cornerstone of asthma pharmacotherapy and, acting via the glucocorticoid receptor (GR), reduce inflammatory gene expression. While this is often attributed to a direct inhibitory effect of the GR on inflammatory gene transcription, corticosteroids also induce the expression of anti-inflammatory genes in vitro. As there are no data to support this effect in asthmatic subjects taking ICS, we have assessed whether ICS induce anti-inflammatory gene expression in subjects with atopic asthma. Bronchial biopsies from allergen-challenged atopic asthmatic subjects taking inhaled budesonide or placebo were subjected to gene expression analysis using real-time reverse transcriptase-PCR for the corticosteroid-inducible genes (official gene symbols with aliases in parentheses): TSC22D3 [glucocorticoid-induced leucine zipper (GILZ)], dual-specificity phosphatase-1 (MAPK phosphatase-1), both anti-inflammatory effectors, and FKBP5 [FK506-binding protein 51 (FKBP51)], a regulator of GR function. Cultured pulmonary epithelial and smooth muscle cells were also treated with corticosteroids before gene expression analysis. Compared with placebo, GILZ and FKBP51 mRNA expression was significantly elevated in budesonide-treated subjects. Budesonide also increased GILZ expression in human epithelial and smooth muscle cells in culture. Immunostaining of bronchial biopsies revealed GILZ expression in the airways epithelium and smooth muscle of asthmatic subjects. Expression of the corticosteroid-induced genes, GILZ and FKBP51, is up-regulated in the airways of allergen-challenged asthmatic subjects taking inhaled budesonide. Consequently, the biological effects of corticosteroid-induced genes should be considered when assessing the actions of ICS. Treatment modalities that increase or decrease GR-dependent transcription may correspondingly affect corticosteroid efficacy.

Abstract 4635: FK506 binding protein 51 is a possible novel tumoral marker

Abstract Very recently, we have identified FK506 binding protein 51 (FKBP51), an immunophilin phisiologically expressed in lymphocytes, as a protein associated with malignant melanoma. Its expression correlates with tumor aggressiveness and is maximal in metastatic lesions. We have also demonstrated that FKBP51 is a factor of resistance to genotoxic agents, including anthracyclins and ionizing radiations, because it promotes NF-kB activation. Aim of the present study was to investigate FKBP51 expression in solid tumors. Overall 60 samples, stored in our Patology section, of the following tumors: breast, colon, lung, pancreas, ovary and prostate (10 for each tumor), and a comparable number of normal tissue samples, were analysed for FKBP51 expression by immunohistochemistry. An intense signal was observed in all prostatic and ovarian adenocarcinomas analyzed, but only in a proportion of pancreatic cancer samples analyzed (mostly, esocrine pancreas tumors). Furthermore, an intense nuclear signal was observed in colon and lung adenocarcinomas. Immunohistochemistry was negative in breast cancer. Normal tissues of the same histotype showed very low or undetectable levels of FKBP51. We also measured FKBP51 mRNA levels in deparaffinized tissues from breast, pancreas, prostate and lung samples, both normal and tumoral, by Realtime PCR. All normal tissues analyzed, except for prostate, displayed mRNA levels lower than those of peripheral blood mononuclear cells (PBL). FKBP51 levels in prostate and PBL were similar. Increases in mRNA levels, from 3 to 30 folds, were found in prostate, esocrine pancreas and lung cancers, compared to normal tissue of the same histotype. Very low FKBP51 mRNA levels were found in breast cancer. In conclusion, our preliminary results show that FKBP51 expression increases in different solid tumors, including ovary, prostate, lung, colon, and esocrine pancreas, in comparison with the normal tissue counterpart. This study support the conclusion that deregulated FKBP51 can play a role in malignant transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4635.

Glucocorticoid Receptor Activates Poised FKBP51 Locus through Long-Distance Interactions

Recent studies have identified FKBP51 (FK506-binding protein 51) as a sensitive biomarker of corticosteroid responsiveness in vivo. In this work, we have elucidated the molecular mechanisms underlying the induction of FKBP51 by the glucocorticoid receptor (GR) in human A549 lung cancer cells showing robust accumulation of FKBP51 mRNA in response to dexamethasone exposure. Our quantitative chromatin immunoprecipitation scans and enhancer activity analyses indicate that activation of the FKBP51 locus by glucocorticoids in vivo is triggered by the loading of GR to enhancers at about 34 kb 5' and about 87 kb 3' of the transcription start site. Interestingly, the region encompassing these enhancers is bordered by CCCTC-binding factor- and cohesin-binding sites. Dexamethasone treatment also decreased the histone density at several regions of the gene, which was paralleled with the occupancy of SWI/SNF chromatin remodeling complexes within the locus. Moreover, silencing of BRM subunit of the SWI/SNF complex blunted the glucocorticoid induction of the locus. The proximal promoter region along with the major intronic enhancer at approximately 87 kb, at which the GR binding peaked, had elevated levels of histone 3 acetylation and H3K4 trimethylation, whereas H3K36 trimethylation more generally marked the gene body and reflected the occupancy of RNA polymerase II. The occurrence of these active chromatin marks within the FKBP51 locus before glucocorticoid exposure suggests that it is poised for transcription in A549 cells. Taken together, these results indicate that the holo-GR is capable of activating transcription and evoking changes in chromatin structure through distant-acting enhancers.