The major histocompatibility class II (MHCII) transactivator (CIITA) has long been known as the master regulator of MHCII genes, which are critical for normal immune function (reviewed in Reith and colleagues and Krawczyk and Reith). CIITA is a non–DNA-binding coactivator that specifically regulates expression of MHCII molecules, with CIITA deficiency leading to the rare human immunodeficiency disease termed bare lymphocyte syndrome. CIITA also regulates expression of genes encoding accessory proteins required for MHCII-restricted antigen presentation, thus CIITA is central to controlling the response to foreign antigens and the maintenance of tolerance.[1, 2] In this issue of the Journal of Bone and Mineral Research, Benasciutti and colleagues demonstrate that CIITA is also a major regulator of osteoclast (OC) differentiation and bone homeostasis.
CIITA controls the expression of MHCII molecules that are displayed on the surface of antigen presenting cells that present peptides to T cells to initiate an adaptive immune response. The major target genes of CIITA include classic MHCII molecules (human leukocyte antigen [HLA]-DR, HLA-DP, and HLA-DQ), but also additional genes utilized for the presentation of antigenic peptides by MHCII molecules including invariant chain HLA-DM and HLA-DO. Factors that activate or inhibit MHCII expression act via the promoters that drive transcription of the MHC2TA gene, which encodes CIITA.[1, 2] Antigen-presenting cells such as dendritic cells (DCs), B cells, and macrophages, as well as activated human T cells and thymic epithelial cells constitutively express MHCII, and MHCII expression is strongly induced on other cell types by interferon γ (IFNγ). This coordinated regulation of MHCII and other genes necessary for its function is quite unique, thus CIITA has been termed the “master regulator” of MHCII and antigen presentation.[1, 2]
The MHCII promoters are characterized by a group of conserved sequence elements (W, X, X2, and Y boxes) to which a stable macromolecular nucleoprotein complex referred to as the MHCII enhanceosome binds. The enhanceosome components are expressed ubiquitously and do not account for the specificity or inducibility of MHCII expression, therefore the “master regulator” of enhanceosome function is transactivation by CIITA.[1, 2] Expression levels of CIITA are responsive to inflammatory stimuli and are critical for regulation of MHCII expression. The ability of CIITA to regulate MHCII and other key genes for antigen presentation has been thought to be the primary function of CIITA. However, recent studies have implicated CIITA in novel roles, including a role in OCs and bone homeostasis.[4, 5]
The study by Benasciutti and colleagues reports that CIITA is expressed in OC precursors, with increased expression in cells from osteoporotic mice. They report two mouse models in which CIITA is overexpressed that show severe osteopenia and increased numbers of OCs. They study transgenic mice overexpressing CIITA systemically and mice with selective CIITA overexpression in bone marrow myeloid cells (a fortuitous finding in mice deficient in the thymic epithelial MHC2TA promoter [pIV–/–]). In mice, overexpression of CIITA systemically or restricted to the myeloid lineage showed the same loss of over 50% in trabecular bone volume/total volume (BV/TV) in both genders. CIITA overexpressing mice showed increased OC number, elevated serum levels of C-terminal telopeptides of collagen type I, yet normal mineral apposition and bone formation rates in vivo.
Knockdown of CIITA in macrophages showed decreased osteoclastogenesis though the bone phenotype of CIITA hypomorphic mice has not yet been examined. CIITA overexpression led to increased OC differentiation in vitro but normal myeloid differentiation in vitro to macrophages and DCs. The mechanism by which CIITA overexpression increases OC formation is shown to be enhanced colony-stimulating factor-1 receptor (c-fms) and receptor activator of NF-κB (RANK) signals. Interestingly, the authors suggest that the effects of CIITA on osteoclastogenesis are not dependent on CIITA regulation of MHCII expression or T cells because there is a lack of similar OC phenotype in MHCII-deficient mice, and in a transgenic mouse lacking a CIITA promoter but with thymic reexpression of CIITA. Thus, the study suggests an intrinsic effect of CIITA expression in OC precursors that is not necessarily dependent on CIITA effects on the MHCII enhanceosome. Given the shared precursor relationship between OCs and DCs (professional antigen-presenting cells), perhaps the shared though different usage of CTIIA should not be so surprising.
CIITA was previously implicated as having an indirect effect on bone during estrogen deficiency–induced bone loss owing to its effects on antigen presentation. In mice, enhanced CIITA expression was demonstrated postovariectomy, which increased MHCII expression and enhanced activation-induced T-cell proliferation. In these studies, T-cell activation was critical for ovariectomy-induced bone loss, and CIITA was shown to be regulated by estrogen in vivo. CIITA regulation was dependent on IFNγ because ovariectomy led to enhanced IFNγ production by T helper (TH) cells, and IFNγ receptor–deficient (IFNγR−/−) mice failed to upregulate CIITA. Regulation of CIITA expression in OC precursors will be of interest to examine during estrogen deficiency and other states of induced bone loss.
Although DCs are the professional antigen-presenting cells of the immune system, OCs have also been shown to be capable of antigen presentation. OCs can uptake soluble antigens and express MHCI and MCHII, as well as costimulatory molecules such as CD80, CD86, and CD40. OCs can present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells in an MHC-restricted fashion. The role of CIITA in antigen presentation in OCs has not yet been examined. However, OCs are much weaker in T-cell activation than DCs and show differences in cytokine expression in response to activation. Thus, OCs and DCs are both capable of regulation of T-cell immunity but with distinct differences.
Interestingly, a 2010 in vitro study by Kim and colleagues showed the opposite effect regarding the effect of CIITA on osteoclastogenesis. Overexpressed CIITA was proposed to decrease osteoclastogenesis by sequestration of the transcription co-factor CBP/p300 (CBP [CREB (cAMP-response element binding protein) Binding Protein]), with decreased transactivation of the transcription factor cfos (cellular FBJ osteosarcoma virus protooncogene) and decreased nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Prior studies also demonstrated conflicting results with CIITA effects on T-cell polarization. Interleukin 4 (IL-4), a T-helper 2 (Th2) cytokine, was shown to be aberrantly upregulated in Th1 cells derived from CIITA-deficient mice. Transfection experiments suggested that CIITA might suppress IL-4 expression by competing with NFAT for binding to the general coactivator CBP (reviewed in LeibundGut-Landmann and colleagues). However, other studies did not show that CIITA represses IL-4 expression in Th2 cells but rather enhanced Th2 bias during CD4+ T-cell activation (reviewed in LeibundGut-Landmann and colleagues). Perhaps these discrepancies in observed effects of CIITA deficiency or overexpression are a result of the unique regulatory mechanism of CIITA. The coactivator function of CIITA is highly dependent on expression of other transcription factors, coactivators, and/or repressors, which may vary with exact conditions of the study or cell type.
Several diseases, including rheumatoid arthritis, multiple sclerosis, and atherosclerosis/myocardial infarction, are associated a specific MHC2TA polymorphism. In these studies the polymorphism was demonstrated to lead to decreased CIITA expression after IFNγ stimulation, but the mechanism involved in disease pathogenesis is not yet understood. Nonetheless, the same polymorphism has been demonstrated to be of importance in a recent osteoporosis/fracture study. The importance of CIITA in regulation of bone homeostasis is highlighted by the demonstration that the CIITA rs3087456(G) allele was protective against incident fractures in elderly women. The observed protective effect of the CIITA rs3087456(G) allele is associated with lower expression of CIITA and MHCII, which is consistent with the finding in mice that overexpression of CIITA promotes osteoclastogenesis.
The maintenance of bone homeostasis is highly regulated by innate immune factors. In this light it is of interest to note that by structure, CIITA is a member of the NOD-LRR (nucleotide-binding oligomerization domain, leucine-rich repeat proteins) or CATERPILLER protein family, which is best known for regulation of innate stimuli such as IL-1 and Toll-like receptors (TLRs) (reviewed in Ting and Davis). NOD-LRR proteins such as NALP 1 or 3 are critical intermediates in the inflammasome pathway that regulate activation of the inflammatory cytokine IL-1. CIITA contains NOD-LRR domains and one isoform of CIITA contains a CARD domain important in protein-protein interactions.[2, 14] The NOD-LRR proteins evolutionarily precede adaptive immunity, raising the possibility that CIITA may have evolved earlier for innate functions. Benasciutti and colleagues propose that because MHC-based allorecognition predates skeletal evolution, bone co-opted the use of CIITA. However, if CIITA has underlying innate functions that are more primitive, it is equally possible that CIITA was actually co-opted for regulation of both MHCII and bone. Understanding how CIITA regulates osteoclastogenesis will be of significant interest, particularly to determine if the MHCII enhanceosome is involved. It will need to be determined if CIITA regulation of osteoclastogenesis requires the NOD-LRR domains and if CIITA can be shown to have other innate regulatory functions.
Polymorphisms of CIITA are now linked to a variety of disease states associated with aging and autoimmunity as well as fracture risk in elderly women.[12, 13] In pathological states, it is likely that inflammatory cytokine upregulation of CIITA plays a role in bone loss during inflammatory and autoimmune disease. Similarly, significant epigenetic regulation of MHC2TA has been demonstrated to alter CIITA expression, which may also influence bone homeostasis. Thus, CIITA may not be as specific a master regulator as was once thought. However, CIITA is a critical regulator with multiple functions in adaptive and innate immunity and should also be seen as an important factor that links the immune and skeletal systems.