Sang Dai Park

Activated Liver X Receptors Stimulate Adipocyte Differentiation through Induction of Peroxisome Proliferator-Activated Receptor γ Expression

ABSTRACT Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.

A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site.

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

The oncoprotein Bcl-3 can facilitate NF-kappa B-mediated transactivation by removing inhibiting p50 homodimers from select kappa B sites.

Previously we have proposed a role for Bcl‐3 in facilitating transactivation through kappa B sites by counteracting the inhibitory effects of bound, non‐transactivating homodimers of the p50 subunit of NF‐kappa B. Such homodimers are abundant for example in nuclei of unstimulated primary T cells. Here we extend the model and provide new evidence which fulfills a number of predictions. (i) Bcl‐3 preferentially targets p50 homodimers over NF‐kappa B heterodimers since the homodimers are completely dissociated from kappa B sites at concentrations of Bcl‐3 which do not affect NF‐kappa B. (ii) Select kappa B sites associate very strongly and stably with p50 homodimers, completely preventing binding by NF‐kappa B. Such kappa B sites are likely candidates for regulation by p50 homodimers and Bcl‐3. (iii) Bcl‐3 and p50 can be co‐localized in the nucleus, a requirement for active removal of homodimers from their binding sites in vivo. (iv) The ankyrin repeat domain of Bcl‐3 is sufficient for the reversal of p50 homodimer‐mediated inhibition, correlating with the ability of this domain alone to inhibit p50 binding to kappa B sites in vitro. Our data support the model that induction of nuclear Bcl‐3 may be required during cellular stimulation to actively remove stably bound p50 homodimers from certain kappa B sites in order to allow transactivating NF‐kappa B complexes to engage. This exact mechanism is demonstrated with in vitro experiments.

Adipocyte Determination- and Differentiation-dependent Factor 1/Sterol Regulatory Element-binding Protein 1c Regulates Mouse Adiponectin Expression

Adiponectin is exclusively expressed in differentiated adipocytes and plays an important role in regulating energy homeostasis, including the glucose and lipid metabolism associated with increased insulin sensitivity. However, the control of adiponectin gene expression in adipocytes is poorly understood. We show here that levels of adiponectin mRNA and protein are reduced in the white adipose tissue of ob/ob and db/db mice and that there is a concomitant reduction of the adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein 1c (SREBP1c) transcription factor. To determine whether ADD1/SREBP1c regulates adiponectin gene expression, we isolated and characterized the mouse adiponectin promoter. Analysis of the adiponectin promoter revealed putative binding sites for the adipogenic transcription factors ADD1/SREBP1c, peroxisomal proliferator-activated receptor γ and CCAAT enhancer-binding proteins. DNase I footprinting and chromatin immunoprecipitation analyses revealed that ADD1/SREBP1c binds in vitro and in vivo to the proximal promoter containing sterol regulatory element (SRE) motifs. A luciferase reporter containing the promoter was transactivated by ADD1/SREBP1c, and introduction of SRE mutations into the construct abolished transactivation. Adenoviral overexpression of ADD1/SREBP1c also elevated adiponectin mRNA and protein levels in 3T3-L1 adipocytes. Furthermore, insulin stimulated adiponectin mRNA expression in adipocytes and augmented transactivation of the adiponectin promoter by ADD1/SREBP1c. Taken together, these data indicate that ADD1/SREBP1c controls adiponectin gene expression in differentiated adipocytes.

Regulation of Swi6/HP1-dependent heterochromatin assembly by cooperation of components of the mitogen-activated protein kinase pathway and a histone deacetylase Clr6.

A study of gene silencing within the mating-type region of fission yeast defines two distinct pathways responsible for the establishment of heterochromatin assembly. One is RNA interference-dependent and acts on centromere-homologous repeats (cenH). The other is a stochastic Swi6 (the fission yeast HP1 homolog)-dependent mechanism that is not fully understood. Here we find that activating transcription factor (Atf1) and Pcr1, the fission yeast bZIP transcription factors homologous to human ATF-2, are crucial for proper histone deacetylation of both H3 and H4. This deacetylation is a prerequisite for subsequent H3 lysine 9 methylation and Swi6-dependent heterochromatin assembly across the rest of the silent mating-type (mat) region lacking the RNA interference-dependent cenH repeat. Moreover, Atf1 and Pcr1 can form complexes with both a histone deacetylase, Clr6, and Swi6, and clr6 mutations affected the H3/H4 acetylation patterns, similar to the atf1 and pcr1 deletion mutant phenotypes at the endogenous mat loci and at the ctt1+ promoter region surrounding ATF/CRE-binding site. These data suggest that Atf1 and Pcr1 participate in an early step essential for heterochromatin assembly at the mat locus and silencing of transcriptional targets of Atf1. Furthermore, a phosphorylation event catalyzed by the conserved mitogen-activated protein kinase pathway is important for regulation of heterochromatin silencing by Atf1 and Pcr1. These findings suggest a role for the mitogen-activated protein kinase pathway and histone deacetylase in Swi6-based heterochromatin assembly.

The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres

Centromeres of fission yeast are arranged with a central core DNA sequence flanked by repeated sequences. The centromere-associated histone H3 variant Cnp1 (SpCENP-A) binds exclusively to central core DNA, while the heterochromatin proteins and cohesins bind the surrounding outer repeats. CHD (chromo-helicase/ATPase DNA binding) chromatin remodeling factors were recently shown to affect chromatin assembly in vitro. Here, we report that the CHD protein Hrp1 plays a key role at fission yeast centromeres. The hrp1Δ mutant disrupts silencing of the outer repeats and central core regions of the centromere and displays chromosome segregation defects characteristic for dysfunction of both regions. Importantly, Hrp1 is required to maintain high levels of Cnp1 and low levels of histone H3 and H4 acetylation at the central core region. Hrp1 interacts directly with the centromere in early S-phase when centromeres are replicated, suggesting that Hrp1 plays a direct role in chromatin assembly during DNA replication.

8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells

8‐Cl‐cyclic adenosine monophosphate (8‐Cl‐cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8‐Cl‐cAMP further, we investigated the effect of 8‐Cl‐cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8‐Cl‐cAMP, albeit with different sensitivity. Among them, SH‐SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8‐Cl‐cAMP was not reproduced by other cAMP analogues or cAMP‐elevating agents, showing that the effect of 8‐Cl‐cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8‐Cl‐cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8‐Cl‐cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8‐Cl‐cAMP‐induced apoptosis is closely related to cell cycle control. In support of this assumption, 8‐Cl‐cAMP‐induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8‐Cl‐cAMP did not induce apoptosis in primary cultured normal cells and non‐transformed cell lines, showing that 8‐Cl‐cAMP‐induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8‐Cl‐cAMP exerts anticancer activity.

PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells

Gap junctional communication during the progression of cell cycle from quiescent G0 to S phase was examined in cultured clone 9 rat liver cells. The transfer of scrape-loaded fluorescent dye was suppressed immediately after the stimulation of cell cycle progression in a synchronized cell population. Northern blot analysis showed that the temporal disturbance of gap junctional communication in cells passing from G0 to S phase did not result from transcriptional down-regulation of connexin 43. It was also found that the PKC inhibitor, calphostin C, was able to restore intercellular communication in serum stimulated cells. Data suggest a control mechanism by PKC mediated phosphorylation in the regulation of gap junction function which is vulnerable to cell cycling. The loss of gap junctional communication correlated with the increased phosphorylation of connexin 43 on serine residues in clone 9 cells. (Mol Cell Biochem 167: 41-49, 1997)

Lymphocyte activation and the family of NF-kappa B transcription factor complexes.

NF-κB is a transcription factor complex known for some time to play a pivotal role in the regulated expression of a large number of genes which are activated during an immune response. Antigen in the context of the appropriate antigen presenting cell (APC) leads to the proliferation of T cells and the expression of factors (predominantly cytokines) from the competent T cells. These factors in turn stimulate various cells involved in an immune response. Both the signal emanating from the initial antigenic encounter and the secondary factor-mediated responses in other cells involve NF-κB, at least in part. For example, NF-κB has been shown to be essential for the regulated expression of the immunoglobulin k light chain (whence the name NF-κB, a nuclear factor binding to the kappa light chain B element), the tumor necrosis factor alpha (TNF-α) and beta (TNF-β), interferon-β, the IL-2 receptor, the IL-6 cytokine, the IL-2 growth factor, GM-CSF, G-CSF, and MHC-Class I, just to name a few immunomodulatory gene products. NF-κB is implicated also in the induction of various acute phase proteins (e.g. angiotensinogen), some transcription factors/oncogenes (e.g. IRF-1 and c-myc) and, most importantly, in the induction of various viruses, including the human immunodeficiency virus (HIV), cytomegalovirus, adenovirus and SV40. NF-κB mediates its effects through so-called κB elements; the consensus sequence for a κB site reads GGGRNNYYCC, but additional variants exist to which NF-κB binds. A variety of agents as well as factors produced by stimulated cells activate NF-κB in the respective target cells; included among these are a large array of T cell mitogens (e.g. lectins, anti CD3 and anti CD2 antibodies, in addition to antigen/APC), the factors TNF-α and TNF-β, IL-1, double-stranded RNA, chemical agents which cause PKC activation, DNA damaging agents and treatments which produce oxygen radicals. In addition, several viruses have been shown to cause NF-κB activation like the HTLV-I and -II viruses, the HSV-1 virus, the HHV-6 virus, the Hepatitis B virus and the adenovirus. These viruses apparently subvert the cellular mechanisms to induce an activation phenotype in cells. The listing given here for agents which activate NF-κB and for genes regulated by NF-κB is only a partial one and is only meant to convey the central role this transcription factor plays during cellular activation and, in particular, during immune activation (for a complete listing and the appropriate References see recent reviews [1, 2]).

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells.

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid‐induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid–treated SH‐SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH‐SY5Y cells in a dose‐ and time‐dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N‐Myc oncoprotein, and increase of GAP‐43 mRNA. Type II protein kinase A activity increased by 1.5‐fold in differentiated SH‐SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIβ and Cα subunits. Since type II protein kinase A and RIIβ have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIβ in SH‐SY5Y cells. The growth of RIIβ‐overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid‐treated cells. Retinoic acid treatment further increased the RIIβ level and further inhibited the growth of RIIβ‐overexpressing cells, showing strong correlation between the level of RIIβ and growth inhibition. However, RIIβ‐overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid–induced growth inhibition through the up‐regulation of RIIβ/type II protein kinase A. J. Cell. Physiol. 182:421–428, 2000.