New Twists in Gene Regulation by Glucocorticoid Receptor: Is DNA Binding Dispensable?

Given the pleiotropic functions of GC, the plethora of molecular mechanisms through which they regulate gene transcription, and their extensive clinical use and importance, it is of great interest and relevance to identify the molecular basis for each of the actions described above. A clever new approach to this important problem is described by Reichardt et al. 1998xReichardt, H.M, Kaestner, K.H, and Tuckermann, J. Cell. 1998; 93: 531–541Abstract | Full Text | Full Text PDF | PubMed | Scopus (757)See all ReferencesReichardt et al. 1998. Using the latest techniques in reversed mouse genetics, the authors have “knocked-in” a point mutation into the GR gene leading to substitution of alanine 458 by a threonine. As described above this substitution results in a dimerization-defective GR (GRdim) that no longer binds cooperatively to palindromic GREs (Heck et al. 1994xHeck, S, Kullman, M, Gast, A, Ponta, H, Rahmsdorf, H.J, Herrlich, P, and Cato, A.C. EMBO J. 1994; 13: 4087–4095PubMedSee all ReferencesHeck et al. 1994). Using liver nuclear extracts, Reichardt et al. verify that GRdim/dim mice no longer express palindromic GRE binding activity. However, they do not exclude the possibility that GRdim can still bind to GRE halfsites. Nevertheless, using transiently transfected embryonic fibroblast cell lines and RNA prepared from livers of untreated and GC-injected mice, they do show that GRdim/dim mice no longer respond to GC with induction of TAT mRNA (Reichardt et al. 1998xReichardt, H.M, Kaestner, K.H, and Tuckermann, J. Cell. 1998; 93: 531–541Abstract | Full Text | Full Text PDF | PubMed | Scopus (757)See all ReferencesReichardt et al. 1998). In addition, there is a large reduction in the inducibility of an MMTV-based reporter. The residual induction is probably due to the fact that GRdim can bind noncooperatively to the multiple GREs present in the MMTV promoter. Given that these are typical GC-inducible genes, it is safe to assume that GRdim/dim mice are defective by-and-large in mounting a positive transcriptional response to GC. However, some genes containing multiple GREs may be weakly induced, and this could be physiologically relevant.As GRdim/dim mice appear relatively normal and healthy under standard laboratory conditions, the most striking outcome of these experiments is that GRE-mediated gene activation is not necessary for development and survival. These results are surprising because the same group had previously generated GR-deficient mice (GR−/−) by inactivating the GR gene (Cole et al. 1995xCole, T.J, Blendy, J.A, Monaghan, A.P, Krieglstein, K, Schmid, W, Aguzzi, A, Fantuzzi, G, Hummler, E, Unsicker, K, and Schutz, G. Genes Dev. 1995; 9: 1608–1621Crossref | PubMedSee all ReferencesCole et al. 1995). Unlike the GRdim/dim mice, GR−/− mice die shortly after birth due to respiratory failure because their lungs have not matured, lacking surfactant proteins. Clearly, GRE-mediated gene induction is not the basis for GC-induced lung maturation. Although GC are used clinically to induce lung maturation and surfactant expression in human fetuses expected to be delivered prematurely, no functional GREs have been described in the promoter region of any surfactant gene. A common defect for GR−/− and GRdim/dim mice is the failure to express gluconeogenic enzymes. While these results indicate that expression of these enzymes is not necessary for viability under standard laboratory conditions, the lesson learned from human patients suffering from acute adrenal crisis suggests that induction of gluconeogenic enzymes may be required for survival under stress. However, the GRdim/dim mice have not yet been subjected to a stress test to examine this important point.In addition to a defect in GRE-mediated gene induction, the GRdim/dim mice are defective in repression of genes that are regulated through nGREs, exhibiting elevated expression of POMC and prolactin, two genes that are controlled by nGREs (17xSakai, D.D, Helms, S, Carlstedt, D.J, Gustafsson, J.A, Rottman, F.M, and Yamamoto, K.R. Genes Dev. 1988; 2: 1144–1154Crossref | PubMedSee all References, 8xDrouin, J, Sun, Y.L, Chamberland, M, Gauthier, Y, De, L.A, Nemer, M, and Schmidt, T.J. EMBO J. 1993; 12: 145–156PubMedSee all References). The only transcriptional regulatory function that is clearly intact in GRdim/dim mice is transcriptional interference. Reichardt et al. demonstrate that interference with AP-1 activity and repression of AP-1-regulated genes, like collagenase and gelatinase, are not affected in cells isolated from GRdim/dim mice. However, the authors have yet to examine this interesting mutant for regulation of NF-κB activity and repression of NF-κB dependent target genes.Given that transcriptional interference is the only regulatory function that remains intact in GRdim/dim mice, the obvious question that arises is which of the physiological and pharmacological actions of GC are mediated through this mechanism. However, the curious reader will have to wait until the GRdim/dim mice are subjected to more tests and tribulations. For instance, it is likely that the anti-inflammatory and immunosuppressive activity of GC is mostly dependent on interference with AP-1 and NF-κB. Yet the only immune function examined in the GRdim/dim mice is GC-induced thymic involution. Clearly, GRdim/dim mice are fully resistant to GC-induced killing of double positive thymocytes, and thus this killing must not necessitate interference with AP-1 activity, but rather is likely to relie on binding of GR to GREs. However, until we know how NF-κB activity is affected in GRdim/dim mice, we will have to refrain from judging whether repression of NF-κB activity is a major contributor to thymocyte killing by GC. In contrast with thymic involution, GC-mediated killing of human lymphomas, which represents another therapeutic use for GC, does not seem to require the activating function of GR (Helmberg et al. 1995xHelmberg, A, Auphan, N, Caelles, C, and Karin, M. EMBO J. 1995; 14: 452–460PubMedSee all ReferencesHelmberg et al. 1995) and may depend on repression of NF-κB-induced survival factors (5xBeg, A.A and Baltimore, D. Science. 1996; 274: 782–784Crossref | PubMed | Scopus (2658)See all References, 13xLiu, Z.-G, Hu, H, Goeddel, D.V, and Karin, M. Cell. 1996; 87: 565–576Abstract | Full Text | Full Text PDF | PubMed | Scopus (1649)See all References). Also, the feedback inhibition of GC secretion mediated via inhibition of corticotropin releasing factor synthesis in the hypothalamus is most likley based on transcriptional interference rather than binding of GR to nGREs, since the circulating levels of GC are normal in the GRdim/dim mice (Reichardt et al. 1998xReichardt, H.M, Kaestner, K.H, and Tuckermann, J. Cell. 1998; 93: 531–541Abstract | Full Text | Full Text PDF | PubMed | Scopus (757)See all ReferencesReichardt et al. 1998). The most interesting question, at least for clinicians and drug developers, is whether GC administration to GRdim/dim mice will result in repression of genes coding for cytokines, chemokines, adhesion molecules, and enzymes that produce inflammatory mediators. It is the repression of such genes that accounts for most of the anti-inflammatory and immunosuppressive activities of GC. Also of great interest would be how GRdim/dim mice respond to various stresses and whether they display the same drop in blood pressure as exhibited by patients suffering from adrenal crisis. As the ability of GC to restore blood pressure in such patients is most likely mediated through inhibition of prostaglandin and leukotriene production (probably through inhibition of cyclooxygenase 2 induction, which is likely to be mediated by interference with AP-1 or NF-κB), it can be expected that as long as GC will exhibit anti-inflammatory activity in GRdim/dim mice, GC should also be able to maintain blood pressure. Thus, GRdim/dim mice may be able to survive acute stress.Although more experiments need to be performed with these valuable mutant mice before one could reach a sweeping conclusion, I find it difficult to refrain from speculation: if GRdim/dim mice can survive stress, maybe the most important functions of GC are not mediated at all through binding of GR to GREs. If this is indeed the case, maybe the most important function of GR is to regulate negatively the activities of AP-1 and NF-κB, two transcription factors whose involvement in inflammatory responses is evolutionary conserved between mammals and insects (Meister et al. 1997xMeister, M, Lemaitre, B, and Hoffmann, J.A. BioEssays. 1997; 19: 1019–1026Crossref | PubMedSee all ReferencesMeister et al. 1997). Thus, it is entirely possible that GC and GR have evolved as negative regulatory devices that prevent overstimulation and misfiring of the innate immune system. If this is true, which function, DNA binding or transcriptional intereference, evolved first?