Here, we generated and analyzed mice lacking the deacetylase HDAC6. In these mice, greatly elevated tubulin acetylation was observed in most tissues. Therefore, this represents the first in vivo mammalian model for tubulin hyperacetylation. Our results demonstrate that HDAC6 is dispensable and that the level of tubulin acetylation is not critical for embryonic development and adult homeostasis.
Because HDAC6 is expressed the most in the testis, we first examined the development and function of this organ. However, histological analysis of the testis revealed no abnormality in HDAC6 knockout mice; furthermore, the testis is also fully functional, as evidenced from the normal fertility of HDAC6-deficient males (Fig. ). Isolation and analysis of spermatogenic cells of different stages revealed that the absence of HDAC6 led to a dramatic increase in tubulin acetylation already at an early developmental stage (pachytene spermatids [Fig. ]), and this showed no impact on the developmentally regulated acetylation of histone H4. Thus, by several criteria, we found that the lack of HDAC6—as well as the resulting tubulin hyperacetylation—does not impair function of the testis.
We next analyzed bone development, as previous studies had indicated a possible function for HDAC6 in this organ. In particular, it had been shown that the bone-specific transcription factor Runx2/Cbfa1 interacts with HDAC6 and recruits it into the nuclei of osteoblasts, thereby repressing transcription of the p21
). This suggests that HDAC6 may also be recruited to other targets of Runx2/CBFA-1 in differentiating osteoblasts to regulate tissue-specific gene expression and that it can contribute to bone development. We found the gross skeletal anatomy to be similar in HDAC6-deficient and wild-type mice. Yet, the metabolically most active bone compartment, the cancellous bone of the tibia metaphysis, displays slightly increased density at both later scanning time points (Fig. ), thus identifying a minor role for HDAC6 in bone biology. Further studies will be required to elucidate the exact mechanism underlying this phenotype and whether it reflects function of HDAC6 in the nucleus (37
) or in the cytoplasm (6
). Nevertheless, the possibility that HDAC6 may control gene transcription by deacetylating histones in some select cases will need further detailed study. In agreement with this possibility, we observed that artificial recruitment of HDAC6 to promoter DNA leads to transcriptional repression, which is dependent on the integrity of the hdac domains (Y. Zhang, unpublished data). This is also in good agreement with our previous findings, showing that purified HDAC6 is able to efficiently deacetylate histones in vitro (41
We also examined lymphoid cell development and function in the absence of HDAC6. We found that in the bone marrow of mice lacking HDAC6, cells representing all the different stages of B-cell differentiation were present at normal numbers; likewise, thymocytes in the thymus were not affected. Furthermore, mature B and T cells were also present in normal numbers in the spleen (Fig. ). Therefore, lymphoid development is normal in the absence of HDAC6. However, when mice were immunized with the T-cell-dependent antigen DNP-KLH, a small but significant reduction in the production of antigen-specific IgGs was observed (Fig. ). Hence, HDAC6 appears to play a role in the immune response; future work will dissect the molecular basis of this phenotype and define its cellular origin. It is interesting to note that recent experiments with human T cells have identified a role for HDAC6 in the formation of the immune synapse (29
). Also, it was shown that lymphocyte chemotaxis is influenced by HDAC6, albeit apparently independently of its deacetylase activity (5
). In addition, infection of CD4+
T cells by human immunodeficiency virus was found to be enhanced upon HDAC6 knockdown (31
). Thus, HDAC6 appears to impinge on the function of the immune system at different levels.
We also analyzed the function of HDAC6 in primary or established MEFs. In these cells, as in the mouse organs, acetylation of tubulin and also Hsp90 was increased (Fig. and ). We quantified the level of tubulin acetylation in fibroblasts and several mouse organs and found it to be spread over a wide range, from close to 100% in brain extracts to as little as a few percent in testis samples (Fig. ). Furthermore, in every case, the HDAC6 knockout sample showed essentially complete acetylation of tubulin. This indicates that cells are remarkably tolerant to very large changes in the degree of tubulin acetylation. Moreover, we found that in MEFs at least HDAC6 contributes all the tubulin deacetylase activity. Treatment with the SirT2 inhibitor nicotinamide did not further increase tubulin acetylation (Fig. ), and in agreement with this, MEFs that are deficient in SirT2 also showed no increase in tubulin acetylation. In this context, it should be mentioned that recently SirT2 was shown to localize, during mitosis, to chromatin and to act as a deacetylase for histone H4 acetylated on lysine 16 (H4K16Ac) (32
Tubulin proteins, the building blocks of microtubules, are subject to several types of evolutionarily conserved posttranslational modifications, including detyrosination, acetylation, generation of Δ2-tubulin, phosphorylation, polyglutamylation, and polyglycylation. Most of these modifications are reversible, and all, except acetylation, occur at the highly variable carboxyl termini of tubulin α and β subunits. Acetylation takes place on lysine 40 of α-tubulin, is mostly associated with stable microtubular structures, such as axonemes, and occurs after microtubule assembly (reviewed in reference 38
). The in vivo role of acetylated microtubules in cells and whole animals remains an important unanswered question, although several approaches have already been tried in some eukaryotic organisms and mammalian cell lines. For example, overexpression of a nonacetylatable α-tubulin variant in Chlamydomonas reinhardtii
) or complete elimination of tubulin acetylation by site-directed mutagenesis of the usually acetylated lysine residue to arginine in Tetrahymena thermophila
) had no observable phenotype. Also, disruption of the HDAC6
gene in mouse embryonic stem cells, which led to highly increased tubulin acetylation levels, did not significantly affect cell proliferation or differentiation of these cells (42
). It has been argued that tubulin acetylation is just downstream of microtubule stabilization. Various experiments have shown that tubulin becomes acetylated after microtubules are stabilized. For example, it was recently shown that integrin regulates the stability of microtubules via focal adhesion kinase (FAK) and Rho signals (24
). The integrin-FAK signaling pathway may facilitate Rho-mDia signaling through GM1, or through a specialized membrane domain containing GM1, to stabilize microtubules in the leading edge of migrating cells, and is thereby involved in the regulation of cell migration and motility. A role for tubulin acetylation in cell motility has also been proposed on the basis that HDAC6 overexpression increased the chemotactic movement of NIH 3T3 cells (14
), whereas chemical inhibition of HDAC6 impaired cell migration, although it did not change microtubules stability (10
). Very recent experiments showed that inhibition of HDAC6 catalytic activity increased the adhesion area of the cell, thus reducing cell migration (30
). We also tested, similarly to Matsumaya et al. (20
), how HDAC activity influences the resistance of the microtubule network to destabilization by demecolcin. However, in our experiments we could not observe an obvious effect, whether HDAC6 activity was inhibited chemically or genetically and using different experimental setups. In the literature, the relationship between tubulin acetylation and the stability of microtubules has not been seen by all investigators, and this may reflect subtle experimental differences. For example, Palazzo et al. (23
) also did not observe that acetylation of the microtubule network led to increased stability. In addition, detyrosination, a tubulin modification which is an established marker of increased microtubule stability, was not found to be elevated in HDAC6-deficient cells (41
). Collectively, these data suggest that in vivo tubulin acetylation is not a direct cause for microtubule stabilization but rather could be one of the consequences of increased microtubule stability. The fact that different groups have reported conflicting results on the importance of tubulin acetylation for microtubule stability suggests that this process is still not entirely understood and is difficult to fully control experimentally. The availability of the HDAC6 knockout mice described here and also of overexpression mouse models (our unpublished data) will be instrumental to better understand the role of tubulin acetylation in vivo.
Similar to what had been seen in human cells following HDAC6 knockdown, we also found that Hsp90 is hyperacetylated in MEFs lacking HDAC6. This results in impaired activation of the GR, reflected in defective nuclear translocation of the receptor upon hormone treatment and reduced activation of a GR-dependent reporter gene (Fig. ), similar to what has been observed in human cells (17
). It will be interesting to examine whether other Hsp90-mediated processes are altered in the absence of HDAC6.
HDAC inhibitors appear very promising for cancer therapy, and their mechanism of action is usually considered to be at the epigenetic level, through an increase in histone acetylation; however, most of the inhibitors used so far do not discriminate between classic HDACs and HDAC6. Therefore, they also lead to an increase in acetylation of tubulin, Hsp90, and possible other substrates, and this will have to be considered for clinical applications. The genetic ablation of HDAC6 presented here suggests that pharmacological inhibition of this enzyme may have only a few side effects, at least in a nonpathological setting. In fact, HDAC6 inhibition has recently been considered as a potential way to induce antitumor activity in multiple myeloma cells (12
). Nevertheless, the precise in vivo contributions of the different HDACs remain to be better defined in normal and pathological situations.