Toll-like receptor expression and functional behavior in platelets from patients with systemic lupus erythematosus.

BACKGROUND: Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. MATERIALS AND METHODS: Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. RESULTS: In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. CONCLUSION: Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model.

Background: CD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. Objectives: We evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. Methods: CD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. Results: CD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. Conclusion: These data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

The Anti-Inflammatory Effect of Nanoarchaeosomes on Human Endothelial Cells.

Archaebacterias are considered a unique source of novel biomaterials of interest for nanomedicine. In this perspective, the effects of nanoarchaeosomes (ARC), which are nanovesicles prepared from polar lipids extracted from the extreme halophilic Halorubrum tebenquinchense, on human umbilical vein endothelial cells (HUVEC) were investigated in physiological and under inflammatory static conditions. Upon incubation, ARC (170 nm mean size, -41 mV ζ) did not affect viability, cell proliferation, and expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin under basal conditions, but reduced expression of both molecules and secretion of IL-6 induced by lypopolysaccharide (LPS), Pam3CSK4 or Escherichia coli. Such effects were not observed with TNF-α or IL-1ß stimulation. Interestingly, ARC significantly decreased basal levels of von Willebrand factor (vWF) and levels induced by all stimuli. None of these parameters was altered by liposomes of hydrogenated phosphatidylcholine and cholesterol of comparable size and concentration. Only ARC were endocytosed by HUVEC and reduced mRNA expression of ICAM-1 and vWF via NF-ĸB and ERK1/2 in LPS-stimulated cells. This is the first report of the anti-inflammatory effect of ARC on endothelial cells and our data suggest that its future use in vascular disease may hopefully be of particular interest.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): Toxicity and elucidation of their anti-inflammatory mechanism(s) of action.

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1ß, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): toxicity and elucidation of their anti-inflammatory mechanism(s) of action

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1β, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.

Oxidative stress induces transcription of telomeric repeat-containing RNA (TERRA) by engaging PKA signaling and cytoskeleton dynamics.

Long non-coding RNAs transcribed from telomeres, known as TERRA (telomeric repeat-containing RNA), are associated with telomere and genome stability. TERRA abundance responds to different cell stresses; however, no studies have focused on oxidative stress, condition that damages biomolecules and is involved in aging and disease. Since telomeres are prone to oxidative damage leading to their dysfunction, our objective was to characterize TERRAs and the mechanisms that control their expression. TERRA increased in cells exposed to H2O2 and reverted by antioxidant treatment. TERRAs are also induced in brown adipose tissue of mice exposed to cold, which raises mitochondrial ROS. In cells exposed to H2O2, ChIP showed that chromatin landscape was modified favoring telomere transcription. TERRAs interacted with HP1α/γ, proteins that were found recruited to subtelomeres. Since HP1γ interacts with the transcriptional machinery, TERRAs may stimulate their own expression by recruiting HP1γ to subtelomeres. TERRA induction reverted within 2 h after removal of H2O2 from culture medium, suggesting they have protective functions. This was supported by rapid TERRA induction following a second H2O2 challenge. PKA inhibitors H89 and PKI blocked TERRA increase by H2O2 or IBMX+Forskolin treatment, suggesting PKA signaling regulates TERRA induction. Treatment of cells with drugs that disturb cytoskeleton integrity or growing cells on surfaces of different stiffness known to generate differential cytoskeleton tension also modified TERRA levels and sensitized cells to lower H2O2 concentrations. In summary, we show that TERRAs are induced in response to oxidative stress and are regulated by PKA as well as by changes in cytoskeleton dynamics.

Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity.

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies.

Leptospira species promote a pro-inflammatory phenotype in human neutrophils.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL-8. In addition, both species increased levels of pro-inflammatory IL-1ß and IL-6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4-blocking antibodies abrogated the IL-8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro-inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis.

Role of neutrophils in CVB3 infection and viral myocarditis.

Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1ß, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.

Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis.

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3-/-) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-ß1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.

Heterochromatin protein (HP)1γ is not only in the nucleus but also in the cytoplasm interacting with actin in both cell compartments.

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.

Organization of nuclear architecture during adipocyte differentiation.

Obesity is a serious health problem worldwide since it is a major risk factor for chronic diseases such as type II diabetes. Obesity is the result of hyperplasia (associated with increased adipogenesis) and hypertrophy (associated with decreased adipogenesis) of the adipose tissue. Therefore, understanding the molecular mechanisms underlying the process of adipocyte differentiation is relevant to delineate new therapeutic strategies for treatment of obesity. As in all differentiation processes, temporal patterns of transcription are exquisitely controlled, allowing the acquisition and maintenance of the adipocyte phenotype. The genome is spatially organized; therefore decoding local features of the chromatin language alone does not suffice to understand how cell type-specific gene expression patterns are generated. Elucidating how nuclear architecture is built during the process of adipogenesis is thus an indispensable step to gain insight in how gene expression is regulated to achieve the adipocyte phenotype. Here we will summarize the recent advances in our understanding of the organization of nuclear architecture as progenitor cells differentiate in adipocytes, and the questions that still remained to be answered.

Adipogenesis is under surveillance of Hsp90 and the high molecular weight Immunophilin FKBP51.

Adipose tissue plays a central role in the control of energy balance as well as in the maintenance of metabolic homeostasis. It was not until recently that the first evidences of the role of heat shock protein (Hsp) 90 and high molecular weight immunophilin FKBP51 have been described in the process of adipocyte differentiation. Recent reports describe their role in the regulation of PPARγ, a key transcription factor in the control of adipogenesis and the maintenance of the adipocyte phenotype. In addition, novel roles have been uncovered for FKBP51 in the organization of the architecture of the nucleus through its participation in the reorganization of the nuclear lamina. Therefore, the aim of this review is to integrate and discuss the recent advances in the field, with special emphasis on the roles of Hsp90 and FKBP51 in the process of adipocyte differentiation.

[The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA]. / La redistribución dinámica mitocondria-núcleo de la inmunofilina FKBP51 es regulada por pka para modular la expresión de genes durante el proceso de adipogénesis.

Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

Dynamic mitochondrial-nuclear redistribution of the immunophilin FKBP51 is regulated by the PKA signaling pathway to control gene expression during adipocyte differentiation.

Glucocorticoids play an important role in adipogenesis through the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90•Hsp70 and one high molecular weight immunophilin, either FKBP51 or FKBP52. When 3T3-L1 preadipocytes are induced to differentiate, FKBP51 expression progressively increases, whereas FKBP52 decreases, and Hsp90, Hsp70, p23 and Cyp40 remain unchanged. Interestingly, FKBP51 rapidly translocates from mitochondria to the nucleus where it is retained upon its interaction with chromatin and the nuclear matrix. FKBP51 nuclear localization is transient, and after 48 hours it cycles back to mitochondria. Importantly, this dynamic FKBP51 mitochondrial-nuclear shuttling depends on PKA signaling, because its inhibition by PKI or knockdown of PKA-cα by siRNA, prevented FKBP51 nuclear translocation induced by IBMX. In addition, the electrophoretic pattern of migration of FKBP51 is altered by treatment of cells with PKI or knockdown of PKA-cα, suggesting that FKBP51 is a PKA substrate. In preadipocytes, FKBP51 colocalizes with PKA-cα in mitochondria. When adipogenesis is triggered, PKA-cα also moves to the nucleus colocalizing with FKBP51 mainly in the nuclear lamina. Moreover, FKBP51 and GR interaction increases when preadipocytes are induced to differentiate. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced FKBP51 nuclear translocation, but not by a specific activator of EPAC. FKBP51 knockdown facilitates adipogenesis, whereas ectopic expression of FKBP51 blocks adipogenesis. These findings indicate that the dynamic mitochondrial-nuclear shuttling of FKBP51 regulated by PKA may be key in fine-tuning the transcriptional control of GR target genes required for the acquisition of adipocyte phenotype.

La redistribución dinámica mitocondria-núcleo de la inmunofilina FKBP51 es regulada por pka para modular la expresión de genes durante el proceso de adipogénesis / [The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

La redistribución dinámica mitocondria-núcleo de la inmunofilina FKBP51 es regulada por pka para modular la expresión de genes durante el proceso de adipogénesis. / [The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

Visualization by BiFC of different C/EBPß dimers and their interaction with HP1α reveals a differential subnuclear distribution of complexes in living cells.

How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPß not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPß dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (Liver Activating Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (Liver Inhibitory Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPß-dependent gene c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPß associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.