The transportosome system as a model for the retrotransport of soluble proteins.

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

Mitochondrial-nuclear communication by FKBP51 shuttling.

The HSP90-binding immunophilin FKBP51 is a soluble protein that shows high homology and structural similarity with FKBP52. Both immunophilins are functionally divergent and often show antagonistic actions. They were first described in steroid receptor complexes, their exchange in the complex being the earliest known event in steroid receptor activation upon ligand binding. In addition to steroid-related events, several pleiotropic actions of FKBP51 have emerged during the last years, ranging from cell differentiation and apoptosis to metabolic and psychiatric disorders. On the other hand, mitochondria play vital cellular roles in maintaining energy homeostasis, responding to stress conditions, and affecting cell cycle regulation, calcium signaling, redox homeostasis, and so forth. This is achieved by proteins that are encoded in both the nuclear genome and mitochondrial genes. This implies active nuclear-mitochondrial communication to maintain cell homeostasis. Such communication involves factors that regulate nuclear and mitochondrial gene expression affecting the synthesis and recruitment of mitochondrial and nonmitochondrial proteins, and/or changes in the functional state of the mitochondria itself, which enable mitochondria to recover from stress. FKBP51 has emerged as a serious candidate to participate in these regulatory roles since it has been unexpectedly found in mitochondria showing antiapoptotic effects. Such localization involves the tetratricopeptide repeats domains of the immunophilin and not its intrinsic enzymatic activity of peptidylprolyl-isomerase. Importantly, FKBP51 abandons the mitochondria and accumulates in the nucleus upon cell differentiation or during the onset of stress. Nuclear FKBP51 enhances the enzymatic activity of telomerase. The mitochondrial-nuclear trafficking is reversible, and certain situations such as viral infections promote the opposite trafficking, that is, FKBP51 abandons the nucleus and accumulates in mitochondria. In this article, we review the latest findings related to the mitochondrial-nuclear communication mediated by FKBP51 and speculate about the possible implications of this phenomenon.

FK506 binding protein 51: Its role in the adipose organ and beyond.

There is a great body of evidence that the adipose organ plays a central role in the control not only of energy balance, but importantly, in the maintenance of metabolic homeostasis. Interest in the study of different aspects of its physiology grew in the last decades due to the pandemic of obesity and the consequences of metabolic syndrome. It was not until recently that the first evidence for the role of the high molecular weight immunophilin FK506 binding protein (FKBP) 51 in the process of adipocyte differentiation have been described. Since then, many new facets have been discovered of this stress-responsive FKBP51 as a central node for precise coordination of many cell functions, as shown for nuclear steroid receptors, autophagy, signaling pathways as Akt, p38 MAPK, and GSK3, as well as for insulin signaling and the control of glucose homeostasis. Thus, the aim of this review is to integrate and discuss the recent advances in the understanding of the many roles of FKBP51 in the adipose organ.

Corticosteroid receptors as a model for the Hsp90•immunophilin-based transport machinery.

Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.

Novel antiadipogenic effect of menadione in 3T3-L1 cells.

Inhibition of adipocyte differentiation can be used as a strategy for preventing adipose tissue expansion and, consequently, for obesity management. Since reactive oxygen species (ROS) have emerged as key modulators of adipogenesis, the effect of menadione (a synthetic form of vitamin K known to induce the increase of intracellular ROS) on 3T3-L1 preadipocyte differentiation was studied. Menadione (15 µM) increased ROS and lipid peroxidation, generating mild oxidative stress without affecting cell viability. Menadione drastically inhibited adipogenesis, accompanied by decreased intracellular lipid accumulation and diminished expression of the lipo/adipogenic markers peroxisome proliferator-activated receptor (PPAR)γ, fatty acid synthase (FAS), CCAAT/enhancer-binding protein (C/EBP) α, fatty acid binding protein (FABP) 4, and perilipin. Menadione treatment also increased lipolysis, as indicated by augmented glycerol release and reinforced by the increased expression of hormone-sensitive lipase (HSL). Additionally, menadione increased the inhibitory phosphorylation of acetyl-CoA-carboxylase (ACC), which results in the inhibition of fatty acid synthesis. As a consequence, triglyceride content was decreased. Menadione also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Further, treatment with increased concentration of insulin, a potent physiological activator of the PI3K/Akt pathway, rescued the normal level of expression of PPARγ, the master regulator of adipogenesis, and overcame the restraining effect of menadione on the differentiation capacity of 3T3-L1 preadipocytes. Our study reveals novel antiadipogenic action for menadione, which is, at least in part, mediated by the PI3K/Akt pathway signaling and raises its potential as a therapeutic agent in the treatment or prevention of adiposity.

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.

Regulation of FKBP51 and FKBP52 functions by post-translational modifications.

FKBP51 and FKBP52 are two iconic members of the family of peptidyl-prolyl-(cis/trans)-isomerases (EC: 5.2.1.8), which comprises proteins that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Originally, both proteins have been studied as molecular chaperones belonging to the steroid receptor heterocomplex, where they were first discovered. In addition to their expected role in receptor folding and chaperoning, FKBP51 and FKBP52 are also involved in many biological processes, such as signal transduction, transcriptional regulation, protein transport, cancer development, and cell differentiation, just to mention a few examples. Recent studies have revealed that both proteins are subject of post-translational modifications such as phosphorylation, SUMOlyation, and acetylation. In this work, we summarize recent advances in the study of these immunophilins portraying them as scaffolding proteins capable to organize protein heterocomplexes, describing some of their antagonistic properties in the physiology of the cell, and the putative regulation of their properties by those post-translational modifications.

Biological Actions of the Hsp90-binding Immunophilins FKBP51 and FKBP52

Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.

Immune response triggered by Trypanosoma cruzi infection strikes adipose tissue homeostasis altering lipid storage, enzyme profile and adipokine expression.

Adipose tissue is a target of Trypanosoma cruzi infection being a parasite reservoir during the chronic phase in mice and humans. Previously, we reported that acute Trypanosoma cruzi infection in mice is linked to a severe adipose tissue loss, probably triggered by inflammation, as well as by the parasite itself. Here, we evaluated how infection affects adipose tissue homeostasis, considering adipocyte anabolic and catabolic pathways, the immune-endocrine pattern and the possible repercussion upon adipogenesis. During in vivo infection, both lipolytic and lipogenic pathways are profoundly affected, since the expression of lipolytic enzymes and lipogenic enzymes was intensely downregulated. A similar pattern was observed in isolated adipocytes from infected animals and in 3T3-L1 adipocytes infected in vitro with Trypanosoma cruzi. Moreover, 3T3-L1 adipocytes exposed to plasmas derived from infected animals also tend to downregulate lipolytic enzyme expression which was less evident regarding lipogenic enzymes. Moreover, in vivo-infected adipose tissue reveals a pro-inflammatory profile, with increased leucocyte infiltration accompanied by TNF and IL-6 overexpression, and adiponectin downregulation. Strikingly, the nuclear factor PPAR-γ is strongly decreased in adipocytes during in vivo infection. Attempts to favor PPAR-γ-mediated actions in the adipose tissue of infected animals using agonists failed, indicating that inflammation or parasite-derived factors are strongly involved in PPAR-γ inhibition. Here, we report that experimental acute Trypanosoma cruzi infection disrupts both adipocyte catabolic and anabolic metabolism secondary to PPAR-γ robust downregulation, tipping the balance towards to an adverse status compatible with the adipose tissue atrophy and the acquisition of an inflammatory phenotype.

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.

NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.

Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.

Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression.

The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms.

[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.

Antiadipogenic effect of carnosic acid, a natural compound present in Rosmarinus officinalis, is exerted through the C/EBPs and PPARγ pathways at the onset of the differentiation program.

BACKGROUND: Obesity is a serious health problem all over the world, and inhibition of adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, inhibits 3T3-L1 preadipocytes differentiation. However, very little is known about the molecular mechanism responsible for its antiadipogenic effect. METHODS: We evaluated the effect of CA on the differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of adipogenic markers, and the subcellular distribution of C/EBPß. RESULTS: CA treatment only during the first day of 3T3-L1 differentiation process was enough to inhibit adipogenesis. This inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPß and C/EBPδ mRNA levels but blocked PPARγ, and FABP4 expression. C/EBPß has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24h of differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPß. CONCLUSIONS: CA exerts its anti-adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPß forms, inducing the loss of C/EBPß proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ. GENERAL SIGNIFICANCE: Understanding the molecular mechanism by which CA blocks adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of obesity.

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.

Management of cytoskeleton architecture by molecular chaperones and immunophilins.

Cytoskeletal structure is continually remodeled to accommodate normal cell growth and to respond to pathophysiological cues. As a consequence, several cytoskeleton-interacting proteins become involved in a variety of cellular processes such as cell growth and division, cell movement, vesicle transportation, cellular organelle location and function, localization and distribution of membrane receptors, and cell-cell communication. Molecular chaperones and immunophilins are counted among the most important proteins that interact closely with the cytoskeleton network, in particular with microtubules and microtubule-associated factors. In several situations, heat-shock proteins and immunophilins work together as a functionally active heterocomplex, although both types of proteins also show independent actions. In circumstances where homeostasis is affected by environmental stresses or due to genetic alterations, chaperone proteins help to stabilize the system. Molecular chaperones facilitate the assembly, disassembly and/or folding/refolding of cytoskeletal proteins, so they prevent aberrant protein aggregation. Nonetheless, the roles of heat-shock proteins and immunophilins are not only limited to solve abnormal situations, but they also have an active participation during the normal differentiation process of the cell and are key factors for many structural and functional rearrangements during this course of action. Cytoskeleton modifications leading to altered localization of nuclear factors may result in loss- or gain-of-function of such factors, which affects the cell cycle and cell development. Therefore, cytoskeletal components are attractive therapeutic targets, particularly microtubules, to prevent pathological situations such as rapidly dividing tumor cells or to favor the process of cell differentiation in other cases. In this review we will address some classical and novel aspects of key regulatory functions of heat-shock proteins and immunophilins as housekeeping factors of the cytoskeletal network.