Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics.

AIMS: To establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. METHODS: HL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA+reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca(2+)measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells. RESULTS: A time-dependent drop in viability was shown after HEDA (P<0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca(2+) peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca(2+) oscillations with a decreased frequency. CONCLUSION: A threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum.

A kinetically trapped intermediate of FK506 binding protein forms in vitro: chaperone machinery dominates protein folding in vivo.

We have characterised the stability, binding and enzymatic properties of three human FK506 binding proteins (FKBP-12) differing only by the length and sequence of their N-terminus. One construct has a short hexa-his tag (6H-FKBP12); the second longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage site; the third has the long fusion tag removed and is essentially native FKBP-12 (cFKBP12). The proteins were purified both under native conditions and also using a refolding protocol. All three natively purified proteins have, within experimental error, the same peptidyl-prolyl isomerase (PPIase) activity (k(cat)/K(m) approximately 1 x 10(6)M(-1)s(-1)), and bind a natural inhibitor, rapamycin, with the same high affinity (K(d) approximately 6 nM). However, refolding of the protein containing the longer tag in vitro results in reduced PPIase activity (the k(cat)/K(m) was reduced from 1 x 10(6)M(-1)s(-1) to 0.81 x 10(6)M(-1)s(-1)) and a 6-fold affinity loss for rapamycin. Addition of both the long and short N-terminal his-tags slows the refolding kinetics of FKBP-12. However, the shorter his-tagged fusion protein regains fully native activity (> or =95%) while the longer regains only approximately 80-85% of native activity. Equilibrium urea denaturation titrations, isothermal titration calorimetry (ITC), analytical gel-filtration, and fluorescence binding data show that this loss of activity is not due to gross misfolding events, but is rather caused by the formation of a stable but subtly misfolded protein that has reduced peptidyl-prolyl isomerase (PPIase) activity and reduced affinity for rapamycin. The difference in behaviour between the in vitro refolded and native forms is due to the dominant role of the cellular chaperone/folding machinery.

A novel endothelin receptor antagonist CPU0213 improves diabetic cardiac insufficiency attributed to up-regulation of the expression of FKBP12.6, SERCA2a, and PLB in rats.

The depressed sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and Ca2+-release channels (ryanodine receptor RyR2) are involved in the diabetic cardiomyopathy. However, an implication of a down-regulation of FK506-binding protein or calstabin-2 (FKBP12.6) is undefined. It was hypothesized that the down-regulation of FKBP12.6 and SERCA2a of the intracellular calcium handling system is closely related to an up-regulated endothelin (ET) system. An ET receptor antagonist CPU0213 is newly discovered and expected to ameliorate cardiac insufficiency which is mediated by the depressed FKBP12.6 and SERCA2a in diabetic rat heart. Diabetes was developed in male Sprague-Dawley rats 8 weeks after an injection of streptozotocin (60 mg/kg IP), and CPU0213 was instituted 30 mg/kg, SC in the last 4 weeks. The assessment of the cardiac function, cardiac calcium handling proteins, endothelin system, and redox enzyme system were conducted. The compromised cardiac function in diabetic rats was accompanied by a significant down-regulation of expression of FKBP12.6 as well as SERCA2a and phospholamban. These were closely linked with an increased ET-1 and up-regulation of endothelin converting enzyme, PropreET1, and inducible nitric oxide synthase mRNA in diabetic cardiomyopathy. After 4-week treatment, CPU0213 was capable to attenuate completely the down-regulated FKBP12.6 and SERCA2a, and up-regulated ET system in association with a recovery of the cardiac insufficiency of diabetic cardiomyopathy.

Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and osterix in ROS 17/2.8 cells.

FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor alpha1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H2O2 (100 microM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10(-9) M) and 1,25 (OH)2D3 (Vitamin D3) (10(-7) M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.

Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling.

Intense angiogenesis proliferation, a histopathological hallmark distinguishing malignant from benign astrocytoma, is vital for tumor progression. Thus, identifying and targeting specific pathways that promote malignant astrocytoma-induced angiogenesis could have substantial therapeutic benefit. Expression profiling of 13 childhood astrocytomas to determine the expression pattern of 133 angiogenesis-related genes revealed that 44 (33%) genes were differentially expressed (17 were overexpressed, and 27 were underexpressed) between malignant high-grade astrocytomas (HGAs) and benign low-grade astrocytomas. Hierarchical clustering and principal components analysis using only the 133 angiogenesis-related genes distinguished HGA from low-grade astrocytoma in 100% of the samples analyzed, as did unsupervised analyses using the entire set of 9198 expressed genes represented on the array, indicating that the angiogenesis-related genes were reliable markers of pathological grade. A striking new finding was significant overexpression of hypoxia-inducible transcription factor (HIF)-2alpha as well as high-level expression of FK506-binding protein (FKBP) 12 by HGA. Furthermore, 9 of 21 (43%) genes overexpressed by HGA were HIF/FKBP-associated genes. This group included the epidermal growth factor receptor (EGFR), which promotes HIF synthesis, as well as insulin-like growth factor-binding protein 2 (IGFBP2), a target gene of HIF activity. Differential protein expression of HIF-2alpha was validated in an independent group of 16 astrocytomas (P = 0.02). We conclude that the EGFR/FKBP12/HIF-2alpha pathway is important in childhood HGA and represents a potential new therapeutic target.

Expression of FKBP12 in benign and malignant vascular endothelium: an immunohistochemical study on conventional sections and tissue microarrays.

FKBP12 is a cytosolic FK506 binding protein that interacts with calcineurin and thereby mediates the immunosuppressive effects of FK506. Because initial immunohistochemical staining showed abundant expression of FKBP12 in vascular endothelial cells, we evaluated whether it could serve as a marker for vascular neoplasms. We performed immunohistochemical staining of conventional sections from formalin-fixed, paraffin-embedded tissue from 59 benign and malignant vascular neoplasms using a polyclonal rabbit antiserum against FKBP12. Western blot analysis of tissue from 6 angiosarcomas showed a single band at 12 kD, consistent with the published molecular weight for the FKBP12 protein. Together, CD31, CD34, and FKBP12 identified all 59 vascular neoplasms in this study. Specificity of immunohistochemical staining was assessed on 1,321 tissues represented on 7 tissue microarrays. All proteins were occasionally expressed in non-vascular tissue. Six of 8 vascular neoplasms represented on the arrays stained for FKBP12, as did normal vessels in numerous cores. The polyclonal antiserum shows comparable sensitivity (94.9%) and specificity (96.5%) to CD34 and CD31 and may be a useful additional marker for vascular differentiation. Because we have evaluated a large number of tissues by tissue microarray, we anticipate that our estimate of the specificity of immunostaining for FKBP12 as a marker for vascular endothelium will be accurate. In addition, our findings may explain the toxic effects of FK506 on vascular endothelium of the kidney.

Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A.

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.