GRα is a nucleocytoplasmic shuttling protein that has the capacity to bi-directionally cross nuclear membranes. It is considered a hormone-dependent transcription factor responsible for regulating a variety of biological pathways. The regulation of GRα nuclear import/export is a key mechanism regulating gene transcription that is either activated or silenced in response to specific extracellular stimuli.
In our previous studies, we have shown that NTM5 cells express endogenous levels of GRα and GRβ36,44,45
; however, we generated a fluorescent protein-GRα construct to more easily evaluate the mechanisms involved in steroid-induced nuclear import of GRα. We used immunofluorescence microscopy to track the movement of RFP-GRα in trabecular meshwork cells in the absence and presence of DEX, a synthetic GC. In the absence of GCs, RFP-GRα localized mainly in the cytoplasm, whereas treatment with DEX resulted in the efficient translocation of RFP-GRα into the nucleus. In addition, the GRα antagonist RU486 also induced RFP-GRα nuclear translocation. Thus, RFP-GRα maintained ligand-dependent cytoplasm to nuclear shuttling, exactly like the wild-type GRα. Surprisingly, RFP-GRα nuclear entry was suppressed at room temperature with the receptor clustering in small round structures outside the nuclear membrane. The requirement for a 37°C temperature for nuclear entry is consistent with a previous study by Moguileswsky and Philibert.37
These authors demonstrated that nuclear uptake of 3
H-DEX was approximately 10-fold higher at 37°C than at 25°C. In our study, the RU486-induced nuclear translocation of RFP-GRα took longer than with DEX. Our observations are also in agreement with the Moguileswsky and Philibert study,37
which demonstrated that the nuclear uptake of 3
H-DEX was greater than 20-fold higher than 3
The cytoskeleton, which includes actin microfilaments, tubulin microtubules, and intermediate filaments, is very important in regulating a variety of cellular functions. To mediate the cellular effects of extracellular and intracellular stimuli, rearrangements of the cytoskeleton are often required. Our current study shows partial localization of α-tubulin and actin with RFP-GRα ( and ), similar to previous studies showing colocalization of these cytoskeletal elements with GRα using immunofluorescence and immunoprecipitation.31,32
Harrell et al.38
have shown that the overexpression of dynamitin (a 50-kDa subunit of the dynein-associated dynactin complex), which competes for the dynein motor with its cargo, inhibited DEX-induced translocation of GRα to the nucleus in 3T3 mouse fibroblasts. The same group reported similar results using geldanamycin or overexpression of the peptidylprolyl isomerase (PPIase) domain fragment of FKBP52.38
In our study, depolymerization of the microtubules by colchicine disrupted the intracellular organization of the microtubule network, but to our surprise failed to inhibit DEX-induced GRα translocation, suggesting that the microtubule track may not be needed for nuclear trafficking in TM cells. Alternatively, the existence of both microtubule-dependent and -independent pathways for GRα nuclear shuttling may be present. Similarly, cytochalasin D treatment failed to block DEX-induced RFP-GRα nuclear translocation, suggesting the presence of a microfilament-independent mechanism for GRα translocation. Also, neither colchicine nor cytochalasin D prevented RU-486–induced nuclear translocation of RFP-GRα (C and F, respectively).
The association of tubulin and actin with GRα may suggest that microfilaments and microtubules play yet unidentified roles, such as depolymerization of microtubules and or microfilaments. For example, Garnier et al. have shown that HSP90 binding to tubulin dimer prevented microtubule polymerization,49
an effect reproduced by FKBP52 protein.50
Both of these proteins are key components of the GRα complex and both inhibited microtubule polymerization.49,50
In addition, Akner et al.51
have shown that the GRα receptor inhibited microtubule polymerization. A number of investigators have shown that the cytoskeleton may not be required for GRα nuclear translocation. Szapary et al.39
reported that microtubules are not required for either the nuclear translocation or biological activity of glucocorticoid receptors, as steroid-induced gene expression was unchanged in the absence of intact microtubules. Vorgias et al.40
confirmed these findings and also showed that the microfilament network is not involved in GRα nuclear translocation in thymocytes. Galigniana et al.41
confirmed the lack of involvement of microtubules, microfilaments, and intermediate filaments in GFP-GRα nuclear translocation by DEX.
Studies on other nuclear receptors have indicated both microtubule-dependent and -independent nuclear translocation. For example, although Campbell et al.52
have shown that the estrogen receptor nuclear translocation was inhibited by microtubule-disrupting agents, while Kalimi et al. reported the lack of an effect.53
The first group used a relatively high concentration of colchicine (100 μM), which may have been toxic and killed the cells. Perrot-Applanat et al.54
reported that the progesterone receptor (PR) nuclear trafficking is microtubule independent. The same group demonstrated that the PR exited the nucleus into the cytoplasm by the administration of energy-depleting drugs and observed reaccumulation of the receptor in the nucleus on removal of the drugs, regardless of whether the cytoskeleton was intact or disrupted. They suggested that the “karyophilic or importin signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.” Finally, HSP90 nuclear translocation appears also to be microtubule and microfilament independent55
; however, it is possible there are other yet unidentified roles for the microtubules/microfilaments in GRα translocation.
Similar to earlier reports, the nuclear translocation in our study was HSP90 dependent because geldanamycin blocked DEX-induced GFP-GRα nuclear translocation. Geldanamycin, a benzoquinone ansamycin that binds to HSP90 and disrupts its function, inhibited DEX-dependent translocation from the cytoplasm to the nucleus. GRα uses at least two distinct pathways for nuclear trafficking: HSP90-dependent (very fast <10 minutes),28
and HSP90-independent (~1 hour)56
pathways. However, in NTM5 cells, GRα nuclear translocation is absolutely HSP90 dependent. Geldanamycin-treated cells showed lower levels of fluorescent RFP-GRα, suggesting degradation of the destabilized complex, which is consistent with previous reports showing proteolysis of GRα by proteasomal degradation following geldanamycin treatment.57,58
We previously showed that the nuclear translocation of endogenous GRβ in primary TM cells is HSP90 dependent, and that inhibition of this nuclear translocation induced the proteasomal degradation of cytosolic GRβ.47
In summary, we have used the RFP-GRα chimera to study cytoplasmic-nuclear translocation in living cells, and this construct behaves like wild-type GRα in that both DEX and RU486 enhance translocation to the nucleus. The translocation of RFP-GRα is inhibited by the inhibitor of the HSP90 chaperone function, geldanamycin; however, whenever NTM5 cells were treated with cytoskeletal disrupting agents, translocation of RFP-GRα proceeded uninterrupted. Although these observations argue against the model in which the RFP-GRα normally moves along cytoskeletal tracts,37
they are consistent with the model suggesting that the transport mechanism is HSP90 chaperone dependent.21–22,35
The exact mechanism for GRα nuclear translocation remains unclear, as inhibitors of both microtubules and microfilaments failed to block the relocalization of GRα from cytosol to nucleus following activation by glucocorticoid. The presence of alternative pathways for nuclear import might facilitate the uninterrupted and efficient translocation of proteins under different physiological conditions where importin-dependent and/or cytoskeleton-dependent pathways may be compromised.59
Although studies presented in the current report investigated the role of cytoskeleton and heat shock proteins and suggested the lack of cytoskeletal involvement, the identity of importins (proteins responsible for nuclear trafficking) involved in GRα translocation in TM is currently unknown. The use of inhibitors or siRNA against different importins (>13 members), exportins (>5 members), and nuclear pore complex subunits, should be carried out to identify the players involved in GRα translocation. Such studies are of great importance because trabecular meshwork cell lines isolated from normal and glaucomatous donors retain their phenotype and behave like parent tissues in that they differ in levels of GRβ and phagocytic activity, and respond differently to GCs.36,48
Therefore, understanding GRα transport could explain some of the differences in responsiveness between normal and glaucomatous TM cells, which will lead to a better understanding of the molecular mechanisms involved in GC-induced ocular hypertension and glaucoma.