Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular localization following heat shock.

The unactivated steroid receptors are chaperoned into a conformation that is optimal for binding hormone by a number of heat shock proteins, including Hsp90, Hsp70, Hsp40, and the immunophilin, FKBP52 (Hsp56). Together with its partner cochaperones, cyclophilin 40 (CyP40) and FKBP51, FKBP52 belongs to a distinct group of structurally related immunophilins that modulate steroid receptor function through their association with Hsp90. Due to the structural similarity between the component immunophilins, FKBP52 and cyclophilin 40, we decided to investigate whether CyP40 is also a heat shock protein. Exposure of MCF-7 breast cancer cells to elevated temperatures (42 degrees C for 3 hours) resulted in a 75-fold increase in CyP40 mRNA levels, but no corresponding increase in CyP40 protein expression, even after 7 hours of heat stress. The use of cycloheximide to inhibit protein synthesis revealed that in comparison to MCF-7 cells cultured at 37 degrees C, those exposed to heat stress (42 degrees C for 3 hours) displayed an elevated rate of degradation of both CyP40 and FKBP52 proteins. Concomitantly, the half-life of the CyP40 protein was reduced from more than 24 hours to just over 8 hours following heat shock. As no alteration in CyP40 protein levels occurred in cells exposed to heat shock, an elevated rate of degradation would imply that CyP40 protein was synthesized at an increased rate, hence the designation of human CyP40 as a heat shock protein. Application of heat stress elicited a marked redistribution of CyP40 protein in MCF-7 cells from a predominantly nucleolar localization, with some nuclear and cytoplasmic staining, to a pattern characterized by a pronounced nuclear accumulation of CyP40, with no distinguishable nucleolar staining. This increase in nuclear CyP40 possibly resulted from a redistribution of cytoplasmic and nucleolar CyP40, as no net increase in CyP40 expression levels occurred in response to stress. Exposure of MCF-7 cells to actinomycin D for 4 hours resulted in the translocation of the nucleolar marker protein, B23, from the nucleolus, with only a small reduction in nucleolar CyP40 levels. Under normal growth conditions, MCF-7 cells exhibited an apparent colocalization of CyP40 and FKBP52 within the nucleolus.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for GA-binding protein (GABP).

Within steroid receptor heterocomplexes the large tetratricopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (Hsp90) and act coordinately with Hsp90 to modulate receptor activity. The reversible nature of the interaction between the immunophilins and Hsp90 suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a 5-kilobase (kb) 5'-flanking region of the human gene and demonstrated that a approximately 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GABP is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells.

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity. We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations in the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent of de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms.

Two structures of cyclophilin 40: folding and fidelity in the TPR domains.

BACKGROUND: The "large immunophilin" family consists of domains of cyclophilin or FK506 binding protein linked to a tetratricopeptide (TPR) domain. They are intimately associated with steroid receptor complexes and bind to the C-terminal domain of Hsp90 via the TPR domain. The competitive binding of specific large immunophilins and other TPR-Hsp90 proteins provides a regulatory mechanism for Hsp90 chaperone activity. RESULTS: We have solved the X-ray structures of monoclinic and tetragonal forms of Cyp40. In the monoclinic form, the TPR domain consists of seven helices of variable length incorporating three TPR motifs, which provide a convincing binding surface for the Hsp90 C-terminal MEEVD sequence. The C-terminal residues of Cyp40 protrude out beyond the body of the TPR domain to form a charged helix-the putative calmodulin binding site. However, in the tetragonal form, two of the TPR helices have straightened out to form one extended helix, providing a dramatically different conformation of the molecule. CONCLUSIONS: The X-ray structures are consistent with the role of Cyclophilin 40 as a multifunctional signaling protein involved in a variety of protein-protein interactions. The intermolecular helix-helix interactions in the tetragonal form mimic the intramolecular interactions found in the fully folded monoclinic form. These conserved intra- and intermolecular TPR-TPR interactions are illustrative of a high-fidelity recognition mechanism. The two structures also open up the possibility that partially folded forms of TPR may be important in domain swapping and protein recognition.

Regulation of the 1b isoform of the plasma membrane calcium pump by 1,25-dihydroxyvitamin D3 in rat osteoblast-like cells.

The first isogene of the plasma membrane calcium pump (PMCA1) is expressed on the apical plasma membrane of osteoblasts, but its regulation by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has not been studied in this cell type. We studied 1,25(OH)2D3 effects on PMCA1 function, protein, messenger RNA (mRNA), and isoform expression in osteoblasts. Of seven rat and human immortalized osteoblast-like cell lines studied, PMCA1 mRNA expression was confirmed in all. Only ROS 17/2.8 cells expressed measurable PMCA1 protein by Western analysis. Immunocytochemistry indicated that PMCA1 was expressed predominantly on the plasma membrane of ROS 17/2.8 cells. The 1,25(OH)2D3 but not 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] treatment of confluent ROS 17/2.8 cells resulted in an approximate 3- to 5-fold dose-dependent increase in PMCA1 expression of message and protein as assessed by Western and Northern analysis and vesicular 45Ca uptake of membrane vesicles. 1,25(OH)2D3 had no effect on PMCA1 posttranscriptional splicing. The 1b isoform of PMCA was expressed under all experimental conditions. 1,25(OH)2D3 favored increased expression of the 5.5 kilobases (kb) over the 7.5-kb PMCA1b transcript, with a 2-fold proportional increase in the smaller transcript relative to the larger transcript evident at the highest dose of 1,25(OH)2D3 studied. The resultant proportional increase in the smaller 5.5-kb transcript may increase mRNA stability and account for the increase in PMCA1b protein and function with 1,25(OH)2D3. These data provide evidence for the role of 1,25(OH)2D3 and PMCA1b in the regulation of calcium transport in bone cells.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas.

PURPOSE: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. METHODS: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. RESULTS: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. CONCLUSIONS: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Protein coregulators that mediate estrogen receptor function.

The recent discovery of estrogen receptor beta as a biological partner with estrogen receptor alpha in mediating the estrogen response has come at precisely the same time as intensive research is revealing the role played by downstream coregulator proteins in linking nuclear hormone receptor activity to general transcription machinery involved in gene transcriptional activation. In what is a rapidly evolving area of research, findings to date have led to a proposed model of hormonal action, in which a receptor activated by estrogen or cell-membrane-derived phosphorylation-dependent signaling pathways promotes recruitment of selected members of the multifunctional steroid receptor coactivator family and the cointegrators, p300/CBP and P/CAF. The intrinsic histone acetylase activity mediated by these coactivator and cointegrator proteins, alters chromatin structure giving rise to increased transcriptional efficiency. On the other hand, antiestrogen-bound receptors favour the assembly of receptor-corepressor complexes containing the sequence-related corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoid and thyroid hormone receptors), localizing histone deacetylase activity to the promoter and leading to transcriptional repression. The model predicts that a change in the balance between corepressor and coactivator expression in favour of coactivators, might result in antiestrogen resistance. Together with available crystal structure data for estrogen- and antiestrogen-bound receptors, these studies have provided valuable insights into events that occur subsequent to receptor interaction with specific DNA sequences and have helped define the molecular basis of estrogen and antiestrogen activity.

The promoter region of the human PMCA1 gene mediates transcriptional downregulation by 1,25-dihydroxyvitamin D(3).

The gene for plasma membrane calcium pump isoform1 (PMCA1) is expressed in calcium-transporting epithelia and bone mesenchymal cells and is upregulated to 1,25-(OH)(2)D(3) in those tissues. A candidate sequence for a vitamin D response element (VDRE) is present within a 1.7-kb promoter region of the human PMCA1 (hPMCA1) gene. We studied hPMCA1 promoter activity in MDBK and ROS 17/2.8 cell lines as PMCA1 mRNA expression is upregulated by 1,25-(OH)(2)D(3) in both. Structural analysis of the putative hPMCA1 VDRE sequence was performed using mobility shift analysis (EMSA) and nuclear extracts from COS-1 cells expressing human VDR (hVDR) and RXRalpha (hRXRalpha). 1,25-(OH)(2)D(3) induced transrepression of the entire 1.7-kb hPMCA1 promoter and of one promoter deletion construct in ROS 17/2.8 cells but not MDBK cells when assayed by luciferase reporter gene assays. Three additional hPMCA1 promoter deletion constructs were unaffected by 1,25-(OH)(2)D(3) in either cell line. While hVDR and hRXRalpha were capable of complexing with a rat osteocalcin DR3 VDRE, EMSA analysis of the potential VDRE from the hPMCA1 gene did not show interaction of either nuclear receptor. Our results indicate tissue-specific sensitivity of the promoter region of the hPMCA1 gene to direct transcriptional downregulation by 1,25-(OH)(2)D(3) and suggest that any positive regulatory VDRE must lie outside of the 1.7-kb core promoter.

Calcitriol upregulates expression and activity of the 1b isoform of the plasma membrane calcium pump in immortalized distal kidney tubular cells.

The plasma membrane calcium pump (PMCA) is ubiquitously expressed in calcium transporting epithelia. PMCA is encoded by four distinct genes (PMCA1-4) which can be further posttranscriptionally modified. PMCA1b is the only isoform of PMCA1 expressed in kidney and intestine. Calcitriol upregulates PMCA protein expression and activity and PMCA1 mRNA expression in the intestine. Calcitriol has a similar effect on kidney distal tubule PMCA activity in vivo but the cellular basis for this effect has not been studied. PMCA expression in Madin-Darby bovine kidney (MDBK) cells, a distal kidney tubule cell line, was compared with a proximal tubule (LLC-PK1) and embryonic (HEK 293) kidney cell line. Only MDBK cells express PMCA1b mRNA and PMCA protein. In MDBK cells, calcitriol increased steady state expression of PMCA1b mRNA and protein and upregulated the functional activity of PMCA on calcium transport to a similar degree. Furthermore, calcitriol enhanced PMCA1b mRNA stability. These data are consistent with in vivo localization studies demonstrating the distal kidney tubule localization of PMCA protein. Furthermore, they indicate that calcitriol is an important regulator of PMCA activity in the kidney distal tubule by a pathway that includes translation and posttranscriptional modification of PMCA1b.

Purification, characterization and crystallization in two crystal forms of bovine cyclophilin 40.

The purification and crystallization of two different crystal forms of the two-domain protein bovine cyclophilin 40 is reported. Tetragonal crystals grown in methyl pentanediol belong to space group P4222 with unit-cell parameters a = 94.5, c = 118.3 A. Long thin needles grown from PEG belong to space group C2 with unit-cell parameters a = 125.71, b = 47.3, c = 74.6 A, beta = 93.90 degrees. The N-terminal 170 amino acids have significant homology with the well characterized human cyclophilin A. The C-terminal domain is largely made up of three copies of the tetratricopeptide repeat motif thought to be involved in mediating protein-protein interactions. Cyclophilins are frequently found as domains in larger multidomain proteins. To date, only X-ray structures of single-domain cyclophilins have been reported, and this work provides the first example of the purification and crystallization of a larger protein containing a cyclophilin domain.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and hop is located in the dimerization domain of Hsp90.

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90beta and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer.

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Approaches to managed care in Germany.

The economic burden of growing health care expenses requires self-sustaining limitations to spending on health care all over the world. Germany is no exception. In the search for solutions different managed care systems are being introduced into the German public and private health care system commencing in 1996. The gatekeeper project of the largest German public health care corporation and the Berlin network project of a competing public health care corporation are described and evaluated. Managed care as a concept of techniques limiting health care spending will thoroughly change the German health care system within the next decade.

Cyclosporin A potentiates estradiol-induced expression of the cathepsin D gene in MCF7 breast cancer cells.

Although the physiological role of the immunophilins cyclophilin-40 and FKBP52 is unknown, their identification as components of the unactivated estrogen receptor has raised the possibility that they might influence receptor activity in response to the binding of immunosuppressants cyclosporin A and FK506, respectively. We have used Northern analysis to determine the influence of cyclosporin A on the expression of the estrogen-inducible cathepsin D gene in human MCF7 breast cancer cells. We report that 1-3 microM cyclosporin A can potentiate cathepsin D mRNA expression by up to 2-fold in cells treated with 10(-12) to 10(-10) M estradiol. A decreased potentiation effect was noted at higher hormone concentrations. Cyclosporin A alone was unable to induce cathepsin D expression and the increased gene activation observed with combined estradiol/cyclosporin A treatment was negated by the antiestrogen ICI 164,384. Our results suggest that the increased potency of estradiol in the presence of cyclosporin A is associated with an enhanced transcriptional activity of the estrogen receptor and support a role for receptor-associated cyclophilin-40 in the activation process.

Cyclophilin 40 (CyP-40), mapping of its hsp90 binding domain and evidence that FKBP52 competes with CyP-40 for hsp90 binding.

The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the N- and C-terminal ends, respectively. Both microdomains are characterized by alpha-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways.

Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli.

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59).

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.