FK-506 and cyclosporin A : immunosuppressive mechanism of action and beyond

FK-506

A : immunosuppressive

and cyclosporin mechanism

of action and beyond

John J. Siekierka Merck

Research Laboratories,

Cyclosporin have

A

found

their new

and

FK-506

widespread

transplantation. mechanism

Research of

action

several

families

diverse

of

immunosuppressive

important

efforts

aimed

these

drugs

transduction

calcineurin.

Inhibition

biochemical

have proved

to be useful

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pathways.

the

addition

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presence

of of

two of the

interact

28, also known

may

In this way, cyclosporin events

defining

components

phosphatase activity

tissue

molecular to

identification as

that

during

of the receptor families

protein

in both

effect

several

A and FK-506 lymphocytic

cell types.

in Immunology

Introduction

in

function

probes of signaling

Opinion

agents

rejection

elucidating

to

phosphatase

processes. and other

Current

at

have,

drugs, some members

downstream

therapeutic graft

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products

with the Ca2+/calmodulin-dependent as

Rahway, New Jersey, USA

in preventing

functions,

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signal

are

use

immunosuppressive gene

and Nolan H. Sigal

1992, 4:54&52

are unresolved in our understanding of action of these immunosuppressive

Over the past few years, investigations into the immunosuppressive mechanism of action of cyclosporin A (CsA) and FK-506 have led to the identification of a unique signal transduction pathway in T cells. Because the inhibition of early-phase gene expression during T-cell activation is generally thought to be responsible for the in vim immunosuppressive activity of these drugs, this pathway has been most intensively studied in the context of the transcriptional regulation of the interleukin (IL)2 gene (reviewed in [lo*]). However, FK-506 and CsA also block neutrophil, basophil and mast cell degranulation, processes which do not require active transcription, with a potency similar to that observed for the inhibition of IL-2 gene expression in T cells [ 2-51. Early studies on the mechanism of action established a strict correlation between those activation pathways inhibited by FK-506 and CsA and the requirement that the drug-sensitive pathway results in a measurable rise in intracellular Ca2+ [6, 71. Therefore, it has been clear for some time that these compounds do not directly alfect the transcriptional ma chinery, but rather inhibit a subset of Ca2+ -associated signal transduction events that are common to several different cell types and differentiation events. These early observations provided the first glimpse at what was recently found to be the apparent target of both CsA and FK-506: the protein phosphatase 2B, which is depen dent on Ca2+ and calmodulin, also known as calcineurin [8**,9**]. In this review, we will discuss the evidence supporting the calcineurin model and point out issues that

Cyclophilin

of the mechanism agents.

and FKBP

FK-506 and CsA bind to distinct families of cellular receptors. The most abundant and well-characterized fam ily members, FK-506 binding protein (FKBP),, and cy clophilin A (CyP-A) (which binds CsA), are localized in the cytosol, are highly conserved and are ubiquitous [1~*,10,11,12*]. The FKBP and CyP families do not exhibit any appreciable homology at the amino aciQ level. However, FKBP,, and CyP-A share similar enzymatic properties and catalyze the cistruns isomerization of peptidyl-prolyl bonds in proteins and peptides (PPIase activity) [ 13-161. CsA and FK-506 are potent inhibitors of their respective receptor’s PPIase activity, an observation which led early on to the proposal that immunosuppression by FK-506 and CsA was a consequence of PPIase inhibition. Although, in general, there was a good correlation between receptor binding and immunosuppression, the identification of analogs of FK-506 or CsA that were not immunosuppressive but were potent PPIase inhibitors suggested that inhibition of this enzyme *as not sufficient to explain the action of the drugs [ 17,18,19**]. The precise function of PPIase activity in the cell is unknown, although it has been speculated that this activity may be important for protein folding in vivo [20,21]. In

Abbreviations CsA-cyclosporin A; CyP-cyclophilin; FKBP-FK-506 NF-AT-nuclear factor of activated T cells; PMA-phorbol 548

@ Current

Biology

binding protein; hsp-heat shock protein; IL-interleukin; myristate acetate; PPlase-peptldyl-prolyl c&tram isomerase Ltd ISSN 0952-7915

FK-506 and cyclosporin

view of this, it is interesting to note that disruption of the genes encoding the major yeast PPIases has no adverse effects on yeast viability, suggesting that these proteins do not play a fundamental role in protein folding [ I2*,22]. Several additional observations reinforce the notion that the interaction of FK-506 and CsA with FKBP and CyP is necessary but not sufficient for inhibition of signal transduction. First, only a fraction of the intracellular FKBP pool needs to be bound to FK-506 to achieve complete inhibition of IL-2 gene expression [le*]. Second, whereas deletion of the major intracellular receptors for FK-506 or CsA is not a lethal event in yeast, the organ ism fails to grow in the presence of the drugs [ 120,221. These observations suggest that a unique entity is formed through the interaction of the drug and its receptor (the FK-506FKBP or CsA-CyP complex) and that this complex can interact with another protein involved in signal transduction. Elucidation of the structures of FKBP and CyP through X-ray crystallography and nuclear magnetic resonance (NMR) techniques indeed demonstrates that the drugs undergo a dramatic alteration in conformation when bound to their receptors [23*-25-l Thus, the drug-receptor complex forms a unique structural entity that is represented by neither the drug or the receptor alone.

Is calcineurin

involved

in T-cell

activation?

The search for a complex between FK-506FKBP (and CsA-CyP) and additional cellular components led to the observation that both FK-SOGFKBP and CsACyP bind to and inactivate the Ca2+/calmodulin-dependent protein phosphatase, calcineurin [ 8**,9**] The interaction of FK-SOGFKBP and CsACyP complexes with calcineurin appears to represent a ‘gain of function’ by FKBP or CyP since under normal cellular conditions (e.g. in the absence of drugs) neither FKBP nor CyP appear to be associated with calcineurin, although a low-affinity as sociation cannot be ruled out. We have characterized the requirements for FK-506FKBP-calcineurin complex formation in vitro and have found, by direct assay of complex formation via high performance liquid chromatography (HPLC) gel filtration, that complex formation between FK-SOGFKBP and calcineurin exhibits an absolute dependence upon calmodulin [26]. We propose that in vivo, calmodulin binding releases the carboxyterminal inhibitory domain from the active site of calcineurin prior to binding of the FK-SOGFKBP complex. Consistent with this hypothesis is the observation that proteolytic removal of the calcineurin inhibitory domain produces a constitutively active 43 k~ calcineurin fragment which binds FK-506-FKBP in the absence of calmodulin [ 27*]. Interestingly, the interaction of FK-506FKBP with calcineurin appears not to require functional PPIase activity [26]. We have constructed an FKBP mutant protein that exhibits essentially wild type FK-506 binding but has greatly reduced PPIase activity (0.01% of wild type). Since

A Siekierka and Sigal

the mutant FKBP exhibits essentially identical calcineurin binding properties, the results suggest that PPIase activity does not play a role in calcineurin complex formation. Two recent studies have provided in vivo evidence irnplicating calcineurin as the drug-sensitive target in IL-2 transcriptional regulation, The first approach utilized di rect measurement of calcineurin phosphatase activity in crude cell lysates in the T-lymphoma cell line JURKAT [ 28**]. Addition of either FK-506 or CsA led to a dosedependent inhibition of calcineurin phosphatase activity, which directly correlated with the level of inhibition of IL-2 production, Second, O’Keefe et al. [29=-l have demonstrated genetically that calcineurin is the critical drug-sensitive target required for the transcription of the IL-2 gene. They co-transfected into JURKAT cells a calcineurin mutant protein, lacking both the autoinhibitory and calmodulin-binding domains, and an IL-2 promoter construct linked to a reporter gene. Normally, JURKAT cells require stimulation with phorbol myristate acetate (PMA) and ionomycin to induce transcription through the IL-2 promoter. O’Keefe hypothesized that since the mutant calcineurin could dephosphorylate proteins in the absence of Ca2+, only PMA would now be required for activation. Indeed, expression of the calcineurin mu tant removed the Ca2+ -dependence of T-cell activation, but under these conditions, FK-506 and CsA still inhibited transcription through the IL-2 promoter. Significantly, in the presence of the calcineurin mutant, a 4.6.fold higher concentration of either drug was required for the inhibition of IL-2 promoter activity. These results directly demonstrate that calcineurin affects the IL-2 promoter in an FK-506/CsAsensitive manner. Since the experiments reported thus far have focused only on IL-2 transcription, it remains to be determined whether calcineurin will play a similar role in other drug-sensitive processes, such as mast cell degranulation. Interestingly, calcineurin has been shown to play a direct role in exocytosis in the prototoon Paramecium [30], an observation that may be of relevance for other cell types. of calcineurin has not, as yet, The in vivo substrate(s) been defined. One model for the site of calcineurin action relies on the observation that a drug-sensitive transcription factor critical for IL-2 gene expression, termed nuclear factor of activated T cells (NF-AT), is composed of two subunits [31-l. One of the subunits is transported from the cytosol to the nucleus upon Tcell activation. Since dephosphorylation can trigger nuclear transport in other cellular systems, it is attractive to speculate that inhibition of calcineurin phosphatase activity may block a similar translocation process in T cells. Alternatively, calcineurin may be part of a phosphotylation/dephosphorylation cascade. In this model, inhibition of calcineurin phosphatase activity may alter the phosphorylation state of many proteins either directly or through an effect on other kinases and phosphatases. For example, since one of the preferred in vitro substrates of calcineurin is the endogenous inhibitor of phosphatase 1 [ 321, FK-506 and CsA may influence the activity of phosphatase 1 as well as calcineurin.

549

550

Transplantation

What functions cyclophilin

do members

families

of the FKBP and

serve?

Although much of our attention has been focused on the most abundant members of the families, FKBP,, and CyP-A, there is a growing list of proteins that interact with FK-506 and CsA. To date, four CyPs and four FKBPs have been identified (reviewed in [l==] >. The findings that some members appear to be membrane-as sociated [ 33, 341 while others display distinct pharmacological specificity [35] provide few clues as to function, but do raise some interesting questions. Which members of the FKBP or CyP family play a role in immunosuppression? Do the same or different family members mediate the toxic side effects of these drugs? Since the evidence implicating calcineurin as the target for FK-506 and CsA is rather compelling, a minimal requirement for a role in immunosuppression would be the interaction of an FKBP or CyP with calcineurin. A 51 kD FKBP (FKBPst) recently isolated from JURKAT cells [ 261, as well as a 15 kD yeast FKBP, fail to interact with calcineurin/calmodulin in the presence of FK-506, suggesting that calcineurin binding may not be a general property of the FKBP family. The universality of this observation awaits the assay of other FKBP family members for calcineurin binding. In contrast, the three members of the human CyP family (CyPs A, B, C) all appear to interact with calcineurin/calmodulin (as CsA complexes) and inhibit its phosphatase activity [8**,9”,27*]. Interestingly, CyP-B, a member of the CyP family which is associated primarily with the endoplasmic reticulum, exhibits the highest a&t&y (as a complex with CsA) for calcineurin [27-l. The ability of all three human CyPs to interact with calcineurin may reflect the similarity in size (17-21 kD) and amino acid identity (65%) of this family compared with the FKBP family numbers which show a greater range of size (12-100 kD) and 50% identity within the FKBP-like domains. One question that remains to be answered is the role of FKBP and CyP in normal cellular metabolism, since it is unlikely that FKBP and CyP associate with calcineurin in the absence of their respective immunosuppressive ligands. A detailed understanding of the normal physiological roles of FKBP and CyP in cells other than lymphocytes may provide understanding into the toxicity exhibited by FK-506 and CsA. An interesting insight into the putative ‘normal’ function of FKBP is the recent demon stration that FKBP,, can be found complexed with the ryanodine receptor in muscle [36**]. The ryanodine rem ceptor constitutes a major Ca2+ channel found in the sarcoplasmic reticulum of muscle, where it functions in excitation-contraction coupling. While the significance of this is not clear, it is tempting to speculate that FKBP PPIase activity plays a role in regulating the conformation of this channel. It may also be significant that, as is the case for T-cell signal transduction, an FKBP is associated with a transduction pathway involved in Ca2+ -mediated signaling. Calcineurin has also been implicated in the regulation of Ca 2+ channels. Dephosphorylation of a dihydropyridine-sensitive Ca2+ channel (or a closely related protein) in mammalian brain appears to inactivate the channel [37]. This mechanism may be related to the

neurological toxicity observed with FK-506 and CsA, particularly since immunosuppression and toxicity appear to be mechanistically related (see below). A 5659 kD protein, heat shock protein (hsp 56), normally found complexed to glucocorticoid and progestin receptors along with hsp90, has been purified from human T cells and characterized; it was found to bind to FK-506 a&-&y matrixes and to contain an FKBP-like domain [ 38*,39*]. This association of an FKBP with the glucocorticoid receptor is intriguing in view of the fact that glucocorticoids alfect transcriptional processes in many different cell types. These observations suggest multiple roles for FKBPs and CyPs in cellular signal transduction, some of which may be affected by FK-506 and CsA.

Toxicity Although the use of CsA and FK-506 has been of enormous benefit in the treatment of renal and liver allograft rejection, these drugs are not without significant clinical side effects. The major toxicities are firstly neurological, which appears to improve with time, and secondly, rem nal, which appears to be progressive. Surprisingly, both compounds have similar side-effect profiles. This raises the question as to whether the drugs’ toxicity and immunosuppressive activities are mechanistically linked. Support for this hypothesis first came from an analysis of CsA analogs [19*-l and, more recently, with an FK-506 analog [40]. In the latter study, an FK-506 analog could act as an antagonist of both the immunosuppressive and toxic effects of the drug. Therefore, FK-506 and CsA inhibit similar signal transduction pathways, perhaps calcineurin-mediated dephosphorylation events, in lymphocytes and in the cells involved in the toxic mamfestations of the drugs. We cannot yet determine whether different FKBPs and CyPs might be responsible for the drug effects in different cell types, since the pharmacological agents used in these studies do not distinguish among the known family members. A thorough appreciation of the physiological role of FKBPs and CyPs in&m phoid and non-lymphoid cells should provide a greater understanding of the non-immunosuppressive and toxic effects of these drugs.

Conclusion The molecular mechanism of action of FK-506 and CsA has become clearer over the past year with the discovery that both drugs act by inhibiting calcineurin phosphatase activity It is also clear that the drugs’ immunosuppressive activity and toxicity profiles are mechanistically linked, implying that FK-506 and CsA inhibit similar transduction pathways, perhaps calcineurin-mediated dephosphorylation events, in lymphocytes and in cells involved in the toxic manifestations of the drugs. Key questions that remain to be answered include the identification of calcineurin substrates involved in transcriptional regulation

FK-506

and the delineation of which of the FKBP and CyP fam iiy members mediate the drugs’ pharmacological effects. With these insights, as well as additional structural information on drug-receptor-calcineurin interaction, the possibility of designing safer and more effective immunosuppressive agents can be realized.

that both the CyPCsA and FKBP-FK-506 phosphatase activity in vitro. 10.

11.

12.

Our thanks to Drs. iarty Peterson, Michael Tocci, and Greg Wiederrecht for critical review of the manuscript,

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38. .

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JJ Siekerka and NH S&al, Department of Immunology Research, Merck Research laboratories, PO HOX 2000, Rahway, New Jersey 07065, IJSA.