Jonathan G. Scammell

Squirrel Monkey Immunophilin FKBP51 Is a Potent Inhibitor of Glucocorticoid Receptor Binding

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent Kd (6.1 nm) and rank-order of steroid displacement of[ 3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel ...Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex.

Intronic hormone response elements mediate regulation of FKBP5 by progestins and glucocorticoids

Abstract Expression of FKBP51, a large molecular weight immunophilin, is strongly enhanced by glucocorticoids, progestins, and androgens. However, the activity of a 3.4-kb fragment of the FKBP51 gene (FKBP5) promoter was only weakly increased by progestin and we show here that it is unresponsive to glucocorticoids and androgens. The entire FKBP5 was scanned for consensus hormone response elements (HREs) using MatInspector. We found that 2 regions of intron E, which are conserved in rat and mouse FKBP5, contain HRE-like sequences with high match scores. Deoxyribonucleic acid fragments (approximately 1 kb in length) containing these regions were amplified and tested in reporter gene assays for steroid responsiveness. One region of intron E of FKBP5 (pIE2) conferred both glucocorticoid and progestin responsiveness to 2 heterologous reporter genes, whereas the other, less-conserved region of intron E (pIE1) was responsive only to progestins. The inclusion of pIE1 upstream of pIE2 (pIE1IE2) enhanced progestin but not glucocorticoid responsiveness. None of the constructs containing intronic sequences was responsive to androgens. Mutation of the putative HREs within pIE1 and pIE2 eliminated hormone responsiveness. Electrophoretic mobility shift assays demonstrated that progesterone receptors (PR) bound to the HRE in pIE1, whereas both PR and glucocorticoid receptors interacted with the HRE in pIE2. These data suggest that distal intronic elements significantly contribute to transcriptional regulation of FKBP5 by glucocorticoids and progestins.

Glucocorticoid resistance in squirrel monkeys results from a combination of a transcriptionally incompetent glucocorticoid receptor and overexpression of the glucocorticoid receptor co-chaperone FKBP51.

Squirrel monkeys have high cortisol compared to Old World primates to compensate for glucocorticoid resistance. Glucocorticoid resistance in squirrel monkeys may result from mutations in the glucocorticoid receptor (GR) that render it less transcriptionally competent, or expression of the co-chaperone FKBP51 that reduces ligand binding. The goal of this study was to reconcile the contribution of each mechanism. Responsiveness of squirrel monkey GR in COS-7 cells was reduced compared to human GR, but induction of GR activity by maximum dexamethasone concentrations was similar. Also, expression of squirrel monkey FKBP51 reduced responsiveness of both squirrel monkey and human GR in T-REx-293 cells. The EC(50) for dexamethasone was 100-fold higher in cells expressing squirrel monkey GR and excess FKBP51 compared to cells expressing only human GR. Effects of FKBP51 expression and treatment with FK506 were also determined in squirrel monkey SQMK-FP cells that naturally express high levels of FKBP51. Overexpression of FKBP51 in SQMK-FP cells had little effect on GR responsiveness, but treatment with FK506 that blocks the effect of FKBP51 increased GR responsiveness. Thus, glucocorticoid resistance in squirrel monkey cells results from both expression of GRs that are less responsive and overexpression of FKBP51 that further reduces GR responsiveness.

Nmi (N‐Myc interactor) inhibits Wnt/β‐catenin signaling and retards tumor growth

We found that the expression levels of N‐Myc interactor (Nmi) were low in aggressive breast cancer cell lines when compared with less aggressive cell lines. However, the lower levels in the aggressive lines were inducible by interferon‐γ (IFN‐γ). Because Nmi has been reported to be a transcription cofactor that augments IFN‐γ induced transcription activity, we decided to test whether Nmi regulates expression of Dkk1, which is also inducible by IFN‐γ. We established stable clones constitutively expressing Nmi in MDA‐MB‐231 (breast) and MDA‐MB‐435 (melanoma) cell lines. Dkk1 was significantly up‐regulated in the Nmi expressing clones concurrent with reduced levels of the critical transcription cofactor of Wnt pathway, β‐catenin. Treatment of the Nmi expressors with blocking antibody to Dkk1 restored β‐catenin protein levels. c‐Myc is a known downstream target of activated β‐catenin signaling. Treatment of Nmi expressors with the proteosome inhibitor MG132, resulted in elevated β‐catenin levels with concomitant elevation of c‐Myc levels. Our functional studies showed that constitutive expression of Nmi reduced the ability of tumor cells for the invasion, anchorage independent growth and tumor growth in vivo. Collectively, the data suggest that overexpression of Nmi inhibits the Wnt/β‐catenin signaling via up‐regulation of Dkk1 and retards tumor growth.

Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.

Organization and function of the FKBP52 and FKBP51 genes.

Best established as components of steroid hormone receptor complexes, it is now clear that the large molecular weight immunophilins, FKBP52 and FKBP51, play important regulatory roles elsewhere in the cell. This review outlines what is known about the organization of the genes, FKBP4 and FKBP5, respectively, encoding these proteins and describes their diverse actions in the nervous system, reproduction, and cancer. The organization of FKBP4 and FKBP5 is very similar among the chordates, and gene expression is influenced by both genetic and epigenetic mechanisms. Recent studies identifying roles of FKBP52 and FKBP51 in the regulation of the microtubule-associated protein tau and microtubule assembly are discussed, as is their interaction with and influence on the transient receptor potential canonical (TRPC) subfamily of ion channel proteins.

Regulation of secretogranin II mRNA in rat neuronal cultures.

The regulation of SgII mRNA expression was investigated in primary cultures of neurons prepared from the hypothalamus and brainstem of 1-day-old rats. The administration of forskolin (FSK) resulted in a time- and dose-dependent increase in SgII mRNA expression, a 9-fold effect within 6 h being achieved with 10 microM FSK, which maximally increased cellular cAMP levels. SgII mRNA levels remained elevated for 24 h. Activation of protein kinase C with 100 nM phorbol 12-myristate 13-acetate (PMA) also increased SgII mRNA expression, although induction with PMA was slower and more moderate (3.8-fold above control after 24 h). Neither 10 microM 1,9-dideoxyforskolin nor 100 nM 4 alpha-phorbol 12,13-didecanoate, inactive analogues of FSK and PMA respectively, had an effect on SgII mRNA. Depolarization of neuronal cultures with 50 mM KCl had a small and variable effect on SgII mRNA levels (1.8-fold above control) in neuronal cultures and did not influence induction with FSK. To investigate whether neuron-like regulation of SgII mRNA expression could be reproduced in PC12 cells, PC12 cells were treated with 100 nM nerve growth factor (NGF) for 7 days prior to challenge with FSK or PMA. Whereas NGF alone modestly increased SgII mRNA expression in PC12 cells (1.8-fold above control), it did not uncover a stimulatory effect of FSK or PMA. These studies indicate that SgII mRNA expression is enhanced by an increase in cellular cAMP and activation of protein kinase C in primary cultures of neurons and emphasize that SgII mRNA is regulated in a cell-specific manner.

Interleukin-8 is a key mediator of FKBP51-induced melanoma growth, angiogenesis and metastasis

Background:FKBP51 is overexpressed in melanoma and impacts tumour cell properties. However, its comprehensive role in melanoma pathogenesis and underlying mechanism(s) remain elusive.Methods:FKBP51 was stably silenced in aggressive melanoma cell lines and its effect examined in vitro and in mouse model. Histological/immunohistochemical analyses were performed to confirm metastasis, angiogenesis and neutrophil infiltration. Gene expression was analyzed by qRT–PCR, immunoblot and/or ELISA. NF-κB transcriptional activity and promoter binding were monitored by luciferase-based promoter-reporter and ChIP assays, respectively. Interleukin (IL)-8 inhibition was achieved by gene silencing or neutralising-antibody treatment.Results:FKBP51 silencing reduced melanoma growth, metastasis, angiogenesis and neutrophil infiltration and led to IL-8 downregulation through NF-κB suppression in cell lines and tumour xenografts. IL-8 inhibition drastically decreased growth, migration and invasiveness of FKPB51-overexpressing cells; whereas its treatment partially restored the suppressed phenotypes of FKBP51-silenced melanoma cells. Interleukin-8 depletion in conditioned medium (CM) of FKBP51-overexpressing melanoma cells inhibited endothelial cell proliferation and capillary-like structure formation, whereas its treatment promoted these effects in endothelial cells cultured in CM of FKBP51-silenced melanoma cells.Conclusions:FKBP51 promotes melanoma growth, metastasis and angiogenesis, and IL-8 plays a key role in these processes. Thus, targeting of FKBP51 or its upstream or downstream regulatory pathways could lead to effective therapeutic strategies against melanoma.

Granins markers of the regulated secretory pathway

The granins are a family of acidic secretory proteins made up of chromogranin A, chromogranin B, and secretogranin II, which exhibit widespread distribution in endocrine and neuronal cells. The numerous potential sites for proteolytic processing have suggested a role for these peptides as prohormones: several potential degradation products of chromogranin A, pancreastatin, and chromostatin have autocrine activity. On the other hand, an intracellular role for the granins is supported by their propensity to aggregate in a low-pH, high-calcium environment such as found in the trans-Golgi network followed by their efficient sorting to the regulated pathway. As a result, the granins are considered markers for the regulated pathway and may play a role in secretory granule formation.