We present three main discoveries. First, we identify a mechanism by which CQ and its analog AQ function as anti-inflammatory drugs by enhancing the activity of glucocorticoids and thereby repressing the transcription of proinflammation cytokines. Second, we demonstrate an inverse relationship between lysosomal function and glucocorticoid signaling, such that conditions that reduce lysosomal functions (CQ or bafilomycin treatment or TFEB knockdown) lead to increased glucocorticoid signaling. Third, we provide evidence that lysosomes are a site of GR degradation and thus control the stability of cytoplasmic GR. These discoveries provide a mechanistic framework for understanding the role of lysosomes and glucocorticoids in the development and homeostasis of the immune system and also provide a rational basis for developing new therapeutics that can be combined with glucocorticoids for treating inflammation and autoimmune diseases.
As major organelles responsible for the degradation and recycling of cellular substrates, lysosomes play critical roles in host cell defense by digesting intracellular and extracellular pathogens (35
). The catabolic functions of lysosomes are required for antigen processing and presentation to the major histocompatibility complex (MHC) class II (37
), which is crucial both for the development of self-tolerance and for the development of autoimmune diseases by immune systems. Endogenous glucocorticoids, like cortisol, through their interaction with GR, also have critical roles in the development and homeostasis of immune systems (38
). In addition, cortisol and related synthetic glucocorticoids are used to treat inflammation and autoimmune diseases because of their potent inhibitory effects on immune systems. Despite the importance of both the lysosomal and the glucocorticoid pathways in regulating the immune system, the functional relationship between these two pathways had not been identified previously.
The inverse relationship between lysosomal and glucocorticoid pathways provides a new perspective in considering their modulation of the immune system. We reason that lysosomal biogenesis and function would expand in response to attacks of infectious agents on the host cells, which should then repress glucocorticoid signaling so that inflammatory cytokines are produced and attract other components of the immune system. In contrast, the demand for lysosomal functions would be reduced after destruction of the pathogens, and an increased sensitivity to glucocorticoid signaling would repress the expression of genes encoding inflammatory cytokines and chemokines. In this situation of infection and clearance, lysosomes not only serve as clearing houses that help host cells to get rid of pathogens, but also serve as signaling hubs that regulate the activity of the immune system through modulation of glucocorticoid activity.
Having established a relationship between lysosomal function and glucocorticoid signaling, we explored mechanisms by which this occurred and found that lysosomes controlled the stability of cytoplasmic GR without affecting its localization. However, we anticipate that stabilization of the receptor may only be part of the mechanism by which lysosomes regulate cellular responsiveness to glucocorticoids. We speculate that some of the potentiation of glucocorticoid signaling by CQ could result from the cellular consequences of the malfunction of lysosomes. The disruption of lysosomal function affects pathways including protein synthesis, degradation, and trafficking. Homeostasis of lysosomes, as well as of autophagosomes, is critical for many physiological and pathological processes that involve autophagy, including the balance of catabolism and anabolism, antigen presentation, bone remodeling, body development, and tumorigenesis (8
). Therefore, it is unlikely that the activation of glucocorticoid signaling due to interference with lysosomal function can be explained by a single signaling event, which may also partially explain the variability in the gene expression response to lysosomal interference. Detailed mechanisms of this dynamic regulation between lysosome function and gluco-corticoid signaling, including how GR is targeted to lysosomes, will be a subject of future investigation.
The identification of a lysosomal pathway for the cytoplasmic degradation of receptors of the nuclear receptor family was unexpected because it is generally established that the ubiquitin-proteasome system is responsible for the rapid clearance of active transcriptional factors (39
), and the autophagy-lysosome pathway is responsible for the clearance of aged organelles and aggregated damaged proteins (8
). However, we found that nuclear-localized GR (ligand-bound) was mainly degraded by a proteasome-dependent process, and cytoplasmic GR was mainly controlled by lysosomes, although we did note cell-specific differences in the relative contributions of these two pathways. These pathways may also contribute to regulate GR activity differently under different circumstances, such as during differentiation or development.
Similar to GR activity, we found that AR and ER activities were also controlled by lysosomes (). Because AR and ER contribute to the development and progression of prostate cancers and breast cancers (41
), the reduction in AR and ER abundance in response to overexpressed TFEB suggests that increasing lysosomal function could be used as a strategy to reduce AR and ER signaling, which may lead to therapeutic applications for prostate cancer and breast cancer.
Given the widespread clinical use of glucocorticoids for treating inflammatory diseases and cancers, our discovery of the synergism between CQ and glucocorticoids has immediate therapeutic implications. Clinical studies of rheumatoid arthritis have shown that combined treatment of CQ with a low dose of glucocorticoid (for example, prednisolone at <15 mg/day) achieved a better effect, in terms of reducing joint destruction and increasing the remission rate, than either agent alone (44
). Our results provide mechanistic insight for this treatment strategy and suggest that combined treatment not only will allow a lower dose of glucocorticoid to be effective but may also achieve therapeutic effects in situations when even a maximal dose of glucocorticoid fails to suppress the inflammation. Our study addresses why the combined treatment has better effects and provides a rational basis for developing new therapeutic applications by leveraging the synergy between glucocorticoids and lysosomal inhibitors.
In summary, we have discovered a mechanism of action for the antiinflammatory effects of CQ and AQ, in which these lysosomal inhibitors synergize with glucocorticoids to mediate transrepression of proinflammatory signals. Although not all of the anti-inflammatory effects of CQ may be mediated by enhancing the immunosuppressive effect of glucocorticoids, our results suggest that this is a major mechanism responsible for the repression of proinflammatory signals at the transcriptional level. Second, we have discovered an inverse relationship between lysosomal biogenesis and function and glucocorticoid signaling, which has both theoretical importance and therapeutic implication. Combined with the established roles of lysosomes in antigen presentation and pathogen clearance, the present work pushes these ubiquitous organelles further to the center of cellular processing of inflammatory responses. Our work opens a new field of opportunity to explore proteins associated with lysosomal pathways as drug targets for treating inflammation, autoimmune diseases, and cancer.