Accumulation of senescent cells in the adrenal gland induces hypersecretion of corticosterone via IL1ß secretion.

Aging progresses through the interaction of metabolic processes, including changes in the immune and endocrine systems. Glucocorticoids (GCs), which are regulated by the hypothalamic-pituitary-adrenal (HPA) axis, play an important role in regulating metabolism and immune responses. However, the age-related changes in the secretion mechanisms of GCs remain elusive. Here, we found that corticosterone (CORT) secretion follows a circadian rhythm in young mice, whereas it oversecreted throughout the day in aged mice >18 months old, resulting in the disappearance of diurnal variation. Furthermore, senescent cells progressively accumulated in the zF of the adrenal gland as mice aged beyond 18 months. This accumulation was accompanied by an increase in the number of Ad4BP/SF1 (SF1), a key transcription factor, strongly expressing cells (SF1-high positive: HP). Removal of senescent cells with senolytics, dasatinib, and quercetin resulted in the reduction of the number of SF1-HP cells and recovery of CORT diurnal oscillation in 24-month-old mice. Similarly, administration of a neutralizing antibody against IL1ß, which was found to be strongly expressed in the adrenocortical cells of the zF, resulted in a marked decrease in SF1-HP cells and restoration of the CORT circadian rhythm. Our findings suggest that the disappearance of CORT diurnal oscillation is a characteristic of aging individuals and is caused by the secretion of IL1ß, one of the SASPs, from senescent cells that accumulate in the zF of the adrenal cortex. These findings provide a novel insight into aging. Age-related hypersecretory GCs could be a potential therapeutic target for aging-related diseases.

Hypoxia induces downregulation of the tumor-suppressive sST2 in colorectal cancer cells via the HIF-nuclear IL-33-GATA3 pathway.

As a decoy receptor, soluble ST2 (sST2) interferes with the function of the inflammatory cytokine interleukin (IL)-33. Decreased sST2 expression in colorectal cancer (CRC) cells promotes tumor growth via IL-33-mediated bioprocesses in the tumor microenvironment. In this study, we discovered that hypoxia reduced sST2 expression in CRC cells and explored the associated molecular mechanisms, including the expression of key regulators of ST2 gene transcription in hypoxic CRC cells. In addition, the effect of the recovery of sST2 expression in hypoxic tumor regions on malignant progression was investigated using mouse CRC cells engineered to express sST2 in response to hypoxia. Our results indicated that hypoxia-dependent increases in nuclear IL-33 interfered with the transactivation activity of GATA3 for ST2 gene transcription. Most importantly, hypoxia-responsive sST2 restoration in hypoxic tumor regions corrected the inflammatory microenvironment and suppressed tumor growth and lung metastasis. These results indicate that strategies targeting sST2 in hypoxic tumor regions could be effective for treating malignant CRC.

Resveratrol blocks retrotransposition of LINE-1 through PPAR α and sirtuin-6.

The retroelement long interspersed element-1 (LINE-1 or L1) comprises about 17% of the human genome. L1 retrotransposition is known to cause genomic instability and related disorders, and resveratrol suppresses this retrotransposition; however, the underlying mechanism is still not elucidated. Recent observations showed that low-molecular-weight compounds might induce L1 retrotransposition through unknown mechanisms. This study aimed to determine polyphenol resveratrol (RV)'s effect on L1-RTP (retrotransposition) in somatic cells. Surprisingly, RV completely blocked L1-RTP. Experiments using the PPARα inhibitor GW6471 or siRNA-mediated PPARα depletion showed that RV-mediated L1-RTP's inhibition depended on peroxisome proliferator-activated receptor α (PPARα). We demonstrated that RV inhibits p38 and cAMP response element binding protein phosphorylation, which are involved in MAPK signaling, and the L1-ORF1 protein's chromatin recruitment. Furthermore, RV increased the expression of sirtuin-6 (SIRT6), which inhibited the activation of L1. The sirtuins family, SIRT1, SIRT6, and SIRT7, but not SIRT3, are involved in RV-mediated inhibition of L1-RTP. Overall, our findings suggest that RV directly modulates PPARα-mediated L1-RTP in somatic cells and that MAPK signaling interacts with SIRT6 closely and may play a role in preventing human diseases such as cancer.

A novel LncRNA PTH-AS upregulates interferon-related DNA damage resistance signature genes and promotes metastasis in human breast cancer xenografts.

Long noncoding RNAs (lncRNAs) are important tissue-specific regulators of gene expression, and their dysregulation can induce aberrant gene expression leading to various pathological conditions, including cancer. Although many lncRNAs have been discovered by computational analysis, most of these are as yet unannotated. Herein, we describe the nature and function of a novel lncRNA detected downstream of the human parathyroid hormone (PTH) gene in both extremely rare ectopic PTH-producing retroperitoneal malignant fibrous histiocytoma and parathyroid tumors with PTH overproduction. This novel lncRNA, which we have named "PTH-AS," has never been registered in a public database, and here, we investigated for the first time its exact locus, length, transcription direction, polyadenylation, and nuclear localization. Microarray and Gene Ontology analyses demonstrated that forced expression of PTH-AS in PTH-nonexpressing human breast cancer T47D cells did not induce the ectopic expression of the nearby PTH gene but did significantly upregulate Janus kinase-signal transducer and activator of transcription pathway-related genes such as cancer-promoting interferon-related DNA damage resistance signature (IRDS) genes. Importantly, we show that PTH-AS expression not only enhanced T47D cell invasion and resistance to the DNA-damaging drug doxorubicin but also promoted lung metastasis rather than tumor growth in a mouse xenograft model. In addition, PTH-AS-expressing T47D tumors showed increased macrophage infiltration that promoted angiogenesis, similar to IRDS-associated cancer characteristics. Although the detailed molecular mechanism remains imperfectly understood, we conclude that PTH-AS may contribute to tumor development, possibly through IRDS gene upregulation.

25(OH)D3 stimulates the expression of vitamin D target genes in renal tubular cells when Cyp27b1 is abrogated.

Recently, it was reported that 25(OH)D3 (25D3) has physiological bioactivity in certain tissues derived from Cyp27b1 knockout mice. To investigate the function of 25D3 in the kidney as an informational crossroad of various calciotropic substances, we employed the CRISPR-Cas9 system to knock out Cyp27b1 in the mouse renal distal tubular mDCT cell line. Unlike the previously reported mice in which Cyp27b1 was targeted systemically, Cyp27b1 knockout mDCT cells did not produce any measurable 1α,25(OH)2D3 (1,25D3) after 25D3 administration. As was seen with treatment of Cyp27b1 knockout mDCT cells with ≥10-8 M of 1,25D3, the administration of 10-7 M of 25D3 translocated the vitamin D3 receptor (VDR) into the nucleus and promoted the expression of the representative 1,25D3-responsive gene Cyp24a1. The exhaustive target gene profiles of 25D3 were similar to those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of the calcium reabsorption-related gene calbindin-D9K, in a way similar to 1,25D3. We also found that 1,25D3 and 25D3 induced the expression of the megalin gene. A chromatin immunoprecipitation assay identified two vitamin D response elements in the upstream region of the megalin gene that seemed to contribute to its expression. Together, we surmise that the ability of 25D3 to stimulate VDR target genes may provide a novel perspective for its role in certain tissues.

DNA damage response induced by Etoposide promotes steroidogenesis via GADD45A in cultured adrenal cells.

Glucocorticoid production is regulated by adrenocorticotropic hormone (ACTH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway in the adrenal cortex, but the changes in steroidogenesis associated with aging are unknown. In this study, we show that cell-autonomous steroidogenesis is induced by non-ACTH- mediated genotoxic stress in human adrenocortical H295R cells. Low-dose etoposide (EP) was used to induce DNA damage as a genotoxic stress, leading to cellular senescence. We found that steroidogenesis was promoted in cells stained with γH2AX, a marker of DNA damaged cells. Among stress-associated and p53-inducible genes, the expression of GADD45A and steroidogenesis-related genes was significantly upregulated. Immunofluorescence analysis revealed that GADD45A accumulated in the nuclei. Metabolite assay using cultured media showed that EP-treated cells were induced to produce and secrete considerable amounts of glucocorticoid. Knockdown of GADD45A using small interfering RNA markedly inhibited the EP-induced upregulation of steroidogenesis-related gene expression, and glucocorticoid production. A p38MAPK inhibitor, but not a PKA inhibitor, suppressed EP-stimulated steroidogenesis. These results suggest that DNA damage itself promotes steroidogenesis via one or more unprecedented non-ACTH-mediated pathway. Specifically, GADD45A plays a crucial role in the steroidogenic processes triggered by EP-stimulated genotoxic stress. Our study sheds new light on an alternate mechanism of steroidogenesis in the adrenal cortex.

Without 1α-hydroxylation, the gene expression profile of 25(OH)D3 treatment overlaps deeply with that of 1,25(OH)2D3 in prostate cancer cells.

Recently, the antiproliferative action of 1,25(OH)2D3 (1,25D3), an active metabolite of vitamin D3, in the management of prostate cancer has been argued rigorously. In this study, we found that at a physiological concentration, 25(OH)D3 (25D3), the precursor of 1,25D3 and an inactive form of vitamin D because of its much weaker binding activity to the vitamin D receptor (VDR) compared with 1,25D3, had a gene expression profile similar to that of 1,25D3 in prostate cancer LNCaP cells. By immunocytochemistry, western blotting, and CYP27B1 and/or VDR knockdown by small interfering RNAs, we found that 10-7 M 25D3, which is within its uppermost physiological concentration in the bloodstream, induced VDR nuclear import and robustly activated its target genes in the virtual absence of CYP27B1 expression. Comprehensive microarray analyses verified 25D3 bioactivity, and we found that 25D3 target gene profiles largely matched those of 1,25D3, while the presence a small subset of 25D3- or 1,25D3-specific target genes was not excluded. These results indicated that 25D3 shares bioactivity with 1,25D3 without conversion to the latter. Metallothionein 2A was identified as a 1,25D3-specific repressive target gene, which might be a prerequisite for 1,25D3, but not 25D3, to exert its anti-proliferative action in LNCaP cells.

An Excess of CYP24A1, Lack of CaSR, and a Novel lncRNA Near the PTH Gene Characterize an Ectopic PTH-Producing Tumor.

Thus far, only 23 cases of the ectopic production of parathyroid hormone (PTH) have been reported. We have characterized the genome-wide transcription profile of an ectopic PTH-producing tumor originating from a retroperitoneal histiocytoma. We found that the calcium-sensing receptor (CaSR) was barely expressed in the tumor. Lack of CaSR, a crucial braking apparatus in the presence of both intraparathyroid and, probably, serendipitous PTH expression, might contribute strongly to the establishment and maintenance of the ectopic transcriptional activation of the PTH gene in nonparathyroid cells. Along with candidate drivers with a crucial frameshift mutation or copy number variation at specific chromosomal areas obtained from whole exome sequencing, we identified robust tumor-specific cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overproduction, which was not observed in other non-PTH-expressing retroperitoneal histiocytoma and parathyroid adenoma samples. We then found a 2.5-kb noncoding RNA in the PTH 3'-downstream region that was exclusively present in the parathyroid adenoma and our tumor. Such a co-occurrence might act as another driver of ectopic PTH-producing tumorigenesis; both might release the control of PTH gene expression by shutting down the other branches of the safety system (e.g., CaSR and the vitamin D3-vitamin D receptor axis).

Intrinsic ubiquitin E3 ligase activity of histone acetyltransferase Hbo1 for estrogen receptor α.

Estrogen receptors (ER) are important transcription factors to relay signals from estrogen and to regulate proliferation of some of breast cancers. The cycling of estrogen-induced DNA binding and ubiquitin-linked proteolysis of ER potentiates ER-mediated transcription. Indeed, several transcriptional coactivators for ER-dependent transcription ubiquitinate ER. Histone acetyltransferase (HAT) Hbo1/KAT7/MYST2, involved in global histone acetylation, DNA replication, transcription, and cellular proliferation, promotes proteasome-dependent degradation of ERα through ubiquitination. However, molecular mechanism for ubiquitination of ERα by Hbo1 is unknown. Here we report the intrinsic ubiquitin E3 ligase activity of Hbo1 toward the ERα. The ligand, estradiol-17ß, inhibited E3 ligase activity of Hbo1 for ERα in vitro, whereas hyperactive ERα mutants from metastatic breast cancers resistant to hormonal therapy, were better substrates for ERα ubiquitination by Hbo1. Hbo1 knock-down caused increase in ERα expression. Hbo1 is another ERα coactivator that ubiquitinates ERα.

Wild-type and specific mutant androgen receptor mediates transcription via 17ß-estradiol in sex hormone-sensitive cancer cells.

We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone-related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF-7 cells. Here, we investigated whether such aberrant ligand-nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/ß or progesterone receptor. Both suppression of PTHrP and activation of prostate-specific antigen genes were observed after independent administration of 17ß-estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr-Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone-dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr-Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild-type [wt]) and AR (Thr-Ala877) were equally responsible for the E2-AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP-AR (wt) and EGFP-AR (Thr-Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2-AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone-sensitive cancer cells.

Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced.

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3ß2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.

Histone acetyltransferase Hbo1 destabilizes estrogen receptor α by ubiquitination and modulates proliferation of breast cancers.

The estrogen receptor (ER) is a key molecule for growth of breast cancers. It has been a successful target for treatment of breast cancers. Elucidation of the ER expression mechanism is of importance for designing therapeutics for ER-positive breast cancers. However, the detailed mechanism of ER stability is still unclear. Here, we report that histone acetyltransferase Hbo1 promotes destabilization of estrogen receptor α (ERα) in breast cancers through lysine 48-linked ubiquitination. The acetyltransferase activity of Hbo1 is linked to its activity for ERα ubiquitination. Depletion of Hbo1 and anti-estrogen treatment displayed a potent growth suppression of breast cancer cell line. Hbo1 modulated transcription by ERα. Mutually exclusive expression of Hbo1 and ERα was observed in roughly half of the human breast tumors examined in the present study. Modulation of ER stability by Hbo1 in breast cancers may provide a novel therapeutic possibility.

Primary polydipsia, but not accumulated ceramide, causes lethal renal damage in saposin D-deficient mice.

Saposin D-deficient (Sap-D(-/-)) mice develop polydipsia/polyuria and die prematurely due to renal failure with robust hydronephrosis. Such symptoms emerged when they were around 3 mo of age. To investigate the pathogenesis of their water mishandling, we attempted to limit water supply and followed sequential changes of physiological and biochemical parameters. We also analyzed renal histological changes at several time points. At 3 mo old just before water restriction challenge was started, their baseline arginine vasopressin level was comparable to the wild-type (WT) level. Twenty-four-hour water deprivation and desamino d-arginine vasopressin administration improved polydipsia and polyuria to certain degrees. However, creatinine concentrations in Sap-D(-/-) mice were significantly higher than those in WT mice, suggesting that some renal impairment already emerged in the affected mice at this age. Renal histological analyses revealed that renal tubules and collecting ducts were expanded after 3 mo old. After 6 mo old, vacuolar formation was observed, many inflammatory cells migrated around the ducts, and epithelial monolayer cells of tubular origin were replaced by plentiful cysts of various sizes. At 10∼12 mo old, severe cystic deformity appeared. On the other hand, 8-mo-long water restriction started at 4 mo old dramatically improved tubular damage and restored once-dampened amount of tubular aquaporin2 protein to the WT level. Furthermore, 10-mo-long water restriction ameliorated their renal function. Remarkably, by continuing water restriction thereafter, overall survival period became comparable with that of the WT. Together, polyuria, devastating renal tubular lesions, and renal failure were ameliorated by the mere 10-mo-long water restriction, which would trigger lethal dehydration if the disease were to be caused by any processes other than primary polydipsia. Our study demonstrates that long-term water restriction surely improved renal histopathological changes leading to prevention of premature death in Sap-D(-/-) mice.

Degradation of p21Cip1 through anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20) ubiquitin ligase complex-mediated ubiquitylation is inhibited by cyclin-dependent kinase 2 in cardiomyocytes.

Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

Systems analysis of ATF3 in stress response and cancer reveals opposing effects on pro-apoptotic genes in p53 pathway.

Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Negative regulation of parathyroid hormone-related protein expression by steroid hormones.

Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.

Age-associated increase in heterochromatic marks in murine and primate tissues.

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1ß) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.

Skp2 promotes adipocyte differentiation via a p27Kip1-independent mechanism in primary mouse embryonic fibroblasts.

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) complex. Genetic ablation of p27(Kip1) in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27(Kip1) by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2(-/-) MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), largely restored lipid accumulation and PPARgamma gene expression in Skp2(-/-) MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27(Kip1) expression.

Distinct roles of GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure overload.

Glycogen synthase kinase-3 (GSK-3) is a master regulator of growth and death in cardiac myocytes. GSK-3 is inactivated by hypertrophic stimuli through phosphorylation-dependent and -independent mechanisms. Inactivation of GSK-3 removes the negative constraint of GSK-3 on hypertrophy, thereby stimulating cardiac hypertrophy. N-terminal phosphorylation of the GSK-3 isoforms GSK-3alpha and GSK-3beta by upstream kinases (e.g., Akt) is a major mechanism of GSK-3 inhibition. Nonetheless, its role in mediating cardiac hypertrophy and failure remains to be established. Here we evaluated the role of Serine(S)21 and S9 phosphorylation of GSK-3alpha and GSK-3beta in the regulation of cardiac hypertrophy and function during pressure overload (PO), using GSK-3alpha S21A knock-in (alphaKI) and GSK-3beta S9A knock-in (betaKI) mice. Although inhibition of S9 phosphorylation during PO in the betaKI mice attenuated hypertrophy and heart failure (HF), inhibition of S21 phosphorylation in the alphaKI mice unexpectedly promoted hypertrophy and HF. Inhibition of S21 phosphorylation in GSK-3alpha, but not of S9 phosphorylation in GSK-3beta, caused phosphorylation and down-regulation of G1-cyclins, due to preferential localization of GSK-3alpha in the nucleus, and suppressed E2F and markers of cell proliferation, including phosphorylated histone H3, under PO, thereby contributing to decreases in the total number of myocytes in the heart. Restoration of the E2F activity by injection of adenovirus harboring cyclin D1 with a nuclear localization signal attenuated HF under PO in the alphaKI mice. Collectively, our results reveal that whereas S9 phosphorylation of GSK-3beta mediates pathological hypertrophy, S21 phosphorylation of GSK-3alpha plays a compensatory role during PO, in part by alleviating the negative constraint on the cell cycle machinery in cardiac myocytes.