Juha M.T. Hyttinen

AMP-activated protein kinase inhibits NF-κB signaling and inflammation: impact on healthspan and lifespan

Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy metabolic homeostasis and thus a major survival factor in a variety of metabolic stresses and also in the aging process. Metabolic syndrome is associated with a low-grade, chronic inflammation, primarily in adipose tissue. A low-level of inflammation is also present in the aging process. There are emerging results indicating that AMPK signaling can inhibit the inflammatory responses induced by the nuclear factor-κB (NF-κB) system. The NF-κB subunits are not direct phosphorylation targets of AMPK, but the inhibition of NF-κB signaling is mediated by several downstream targets of AMPK, e.g., SIRT1, PGC-1α, p53, and Forkhead box O (FoxO) factors. AMPK signaling seems to enhance energy metabolism while it can repress inflammatory responses linked to chronic stress, e.g., in nutritional overload and during the aging process. AMPK can inhibit endoplasmic reticulum and oxidative stresses which are involved in metabolic disorders and the aging process. Interestingly, many target proteins of AMPK are so-called longevity factors, e.g., SIRT1, p53, and FoxOs, which not only can increase the stress resistance and extend the lifespan of many organisms but also inhibit the inflammatory responses. The activation capacity of AMPK declines in metabolic stress and with aging which could augment the metabolic diseases and accelerate the aging process. We will review the AMPK pathways involved in the inhibition of NF-κB signaling and suppression of inflammation. We also emphasize that the capacity of AMPK to repress inflammatory responses can have a significant impact on both healthspan and lifespan.

Maturation of autophagosomes and endosomes: A key role for Rab7

Macroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimers disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases.

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

The pathogenesis of age‐related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE‐19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG‐132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin‐protein conjugates and the lysosomal membrane protein LAMP‐2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

Endoplasmic Reticulum Stress in Age-Related Macular Degeneration: Trigger for Neovascularization

Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chronic, overwhelming stress that can cause apoptotic cell death. Interestingly, ER stress is a well-known inducer of angiogenesis in cancer. Moreover, stress conditions associated with the progress of AMD can induce the expression of VEGF. We discuss the role of ER stress in the regulation of neovascularization and the conversion of dry AMD to its wet, detrimental counterpart.

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3

Background: HAS isoenzymes differ in enzymatic activity and regulation. Results: HAS1 requires higher UDP-sugar concentration than HAS2 and HAS3. Conclusion: HAS1 activity is highly dependent, and its expression correlates with cellular UDP-sugar supply. Significance: Enhanced UDP-sugar levels are potential mediators of enhanced hyaluronan secretion in cancer and inflammation. Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1–3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1–3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.

Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation diseases.

Processing of misfolded proteins is important in order for the cell to maintain its normal functioning and homeostasis. Three systems control the quality of proteins: chaperone-mediated refolding, proteasomal degradation of ubiquitinated proteins, and finally, when the two others fail, aggrephagy, as selective form of autophagy, degrades ubiquitin-labelled aggregated cargos. In this route misfolded proteins gradually form larger aggregates, aggresomes and they eventually become double membrane-wrapped organelles called autophagosomes, which become degraded when they fuse to lysosomes, for reuse by the cell. The stages, the main molecules participating in the process, and the regulation of aggrephagy are discussed here, as is the role of protein aggregation in protein accumulation diseases. In particular, we emphasize that both Alzheimers disease and age-related macular degeneration, two of the most common pathologies in the aged, are characterized by altered protein clearance and deposits. Based on the hypothesis that manipulations of autophagy may be potentially useful in these and other aggregation-related diseases, we will discuss some promising therapeutic strategies to counteract protein aggregates-induced cellular toxicity.

Context-Dependent Regulation of Autophagy by IKK-NF-κB Signaling: Impact on the Aging Process.

The NF-κB signaling system and the autophagic degradation pathway are crucial cellular survival mechanisms, both being well conserved during evolution. Emerging studies have indicated that the IKK/NF-κB signaling axis regulates autophagy in a context-dependent manner. IKK complex and NF-κB can enhance the expression of Beclin 1 and other autophagy-related proteins and stimulate autophagy whereas as a feedback response, autophagy can degrade IKK components. Moreover, NF-κB signaling activates the expression of autophagy inhibitors (e.g., A20 and Bcl-2/xL) and represses the activators of autophagy (BNIP3, JNK1, and ROS). Several studies have indicated that NF-κB signaling is enhanced both during aging and cellular senescence, inducing a proinflammatory phenotype. The aging process is also associated with a decline in autophagic degradation. It seems that the activity of Beclin 1 initiation complex could be impaired with aging, since the expression of Beclin 1 decreases as does the activity of type III PI3K. On the other hand, the expression of inhibitory Bcl-2/xL proteins increases with aging. We will review the recent literature on the control mechanisms of autophagy through IKK/NF-κB signaling and emphasize that NF-κB signaling could be a potent repressor of autophagy with ageing.

Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER-Golgi-plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.