Gene expression and characterization of clonally derived murine embryonic brown and brite adipocytes.

White adipocytes store energy, while brown and brite adipocytes release heat via nonshivering thermogenesis. In this study, we characterized two murine embryonic clonal preadipocyte lines, EB5 and EB7, each displaying unique gene marker expression profiles. EB5 cells differentiate into brown adipocytes, whereas EB7 cells into brite (also known as beige) adipocytes. To draw a comprehensive comparison, we contrasted the gene expression patterns, adipogenic capacity, as well as carbohydrate and lipid metabolism of these cells to that of F442A, a well-known white preadipocyte and adipocyte model. We found that commitment to differentiation in both EB5 and EB7 cells can be induced by 3-Isobutyl-1-methylxanthine/dexamethasone (Mix/Dex) and staurosporine/dexamethasone (St/Dex) treatments. Additionally, the administration of rosiglitazone significantly enhances the brown and brite adipocyte phenotypes. Our data also reveal the involvement of a series of genes in the transcriptional cascade guiding adipogenesis, pinpointing GSK3ß as a critical regulator for both EB5 and EB7 adipogenesis. In a developmental context, we observe that, akin to brown fat progenitors, brite fat progenitors make their appearance in murine development by 11-12 days of gestation or potentially earlier. This result contributes to our understanding of adipocyte lineage specification during embryonic development. In conclusion, EB5 and EB7 cell lines are valuable for research into adipocyte biology, providing insights into the differentiation and development of brown and beige adipocytes. Furthermore, they could be useful for the characterization of drugs targeting energy balance for the treatment of obesity and metabolic diseases.

FAM129B is a cooperative protein that regulates adipogenesis.

FAM129B is one of Niban-like proteins described in neoplastic cells and implicated in melanoma cell invasion, but no reports have been published on FAM129B and cell differentiation. We show that FAM129B is early and transiently expressed and crucial for 3T3-F442A adipogenesis. Fam129b is expressed downstream of the early genes Cebpb, Klf4, Klf5 and Srebf1a, but upstream of Pparg2 since knockdown of Fam129b blocked Pparg2 expression and adipose differentiation. Glycogen synthase kinase 3 beta activity, a crucial kinase for adipogenesis, and the ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. Phosphorylated FAM129B is crucial for Pparg2 expression and the lipogenic gene expression downstream of Pparg2, and hence for adipogenesis. Fam129b knockdown reduced adipocyte cluster formation and size, regulating commitment and clonal amplification. In vivo, BAT, inguinal and epidydimal fat expressed Fam129b, suggesting a role in adipose tissue development. We conclude that FAM129B is a cooperative protein that regulates differentiation during the early stages of adipogenesis.

A cellular perspective of adipogenesis transcriptional regulation.

Adipose cells store lipids in the cytoplasm and signal systemically through secretion of adipokines and other molecules that regulate body energy metabolism. Differentiation of fat cells and its regulation has been the focus of extensive research since the early 1970s. In this review, we had attempted to examine the research bearing on the control of adipose cell differentiation, some of it dating back to the early days when Howard Green and his group described the preadipocyte cell lines 3T3-L1 and 3T3-F442A during 1974-1975. We also concentrated our attention on research published during the last few years, emphasizing data described on transcription factors that regulate adipose differentiation, outside of those that were reported earlier as part of the canonical adipogenic transcriptional cascade, which has been the subject of ample reviews by several groups of researchers. We focused on the studies carried out with the two preadipocyte cell culture models, the 3T3-L1 and 3T3-F442A cells that have provided essential data on adipose biology.

Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments.

Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

Lipogenic Enzymes Complexes and Cytoplasmic Lipid Droplet Formation During Adipogenesis.

Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.

Retinoic Acid Inhibits Adipogenesis Modulating C/EBPß Phosphorylation and Down Regulating Srebf1a Expression.

Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3ß-C/EBPß-srebf1a axis is a critical signaling pathway at early stages leading to the expression of PPARγ2, the master regulator of adipose differentiation. Previous work has demonstrated that retinoic acid inhibits adipogenesis affecting different signaling pathways. Here, we evaluated the anti-adipogenic effect of retinoic acid on the adipogenic transcriptional cascade, and the expression of adipogenic genes cebpb, srebf1a, srebf1c, pparg2, and cebpa. Our results demonstrate that retinoic acid blocks adipose differentiation during commitment, returning cells to an apparent non-committed state, since they have to be newly induced to adipose conversion after the retinoid is removed from the culture medium. Retinoic acid down regulates the expression of the adipogenic genes, srebf1a, srebf1c, pparg2, and cebpa; however, it did not down regulate the expression of cebpb, but it inhibited C/EBPß phosphorylation at Thr188, a critical step for the progression of the adipogenic program. We also found that RA inhibition of adipogenesis did not increase the expression of dlk1, the gene encoding for Pref1, a well-known anti-adipogenic factor.

The transient expression of Klf4 and Klf5 during adipogenesis depends on GSK3ß activity.

Adipogenesis is regulated by a complex cascade of transcriptional factors, among them KLF4. This factor was previously shown to be necessary for adipose differentiation. We found that GSK3ß activity was required for Klf4 and Klf5 expression during adipogenesis. In addition, retinoic acid inhibited Klf4 and Klf5 expression but not that of Cebpb. Protein synthesis inhibition showed that the transient expression of Klf4, Cebpb and Klf5 during early adipogenesis seemed to require a yet unknown protein for their repression. We also found that Klf4 forced expression in 3T3-F442A cells cultured under non-adipogenic conditions did not induce adipogenesis, nor the expression of Cebpb or Klf5, a Cebpb target gene, showing that KLF4 was not sufficient for adipose differentiation to take place. This would suggest that a more complex combination of molecular pathways not yet understood, is involved during early adipogenesis.

Tissue alkaline phosphatase is involved in lipid metabolism and gene expression and secretion of adipokines in adipocytes.

BACKGROUND: Alkaline phosphatases are dimeric hydrolytic enzymes that dephosphorylate nucleotides and proteins. AP-TNAP is found primarily in skeletal tissues were it plays a major role in the mineralization of the extracellular matrix and bone formation. METHODS: In this study we found through conventional and real time PCR assays that Alpl, the gene encoding for AP-TNAP is expressed in adipose tissue and in 3 T3-F442A adipocytes. We evaluated, using RNAi its role in adipocyte metabolism, and its cytoplasmic location by immunohistochemistry. RESULTS: Alpl is highly expressed late in adipogenesis during adipose terminal differentiation. Knocking down Alpl increased the expression of the genes encoding for glycerophosphate dehydrogenase, and for the adipokines adiponectin, and FABP4 (aP2) but decreased that of leptin, and it also increased secretion of FABP4; these 3 proteins are important in adipocyte systemic signaling and insulin sensitivity. Inhibition of alkaline phosphatase activity in adipocytes by levamisole reduced lipolysis and the expression of various lipogenic genes. We found the enzyme intracytoplasmically, forming aggregates in close surroundings of the lipid droplets during lipolysis. CONCLUSIONS: We suggest that AP-TNAP activity is involved in lipid and energy metabolism of fat cells, and it might regulate glucose metabolism and insulin sensitivity via adipokine synthesis and secretion. GENERAL SIGNIFICANCE: The activity of AP-TNAP might have a critical role in the energy balance of the adipocyte, probably participating in obesity and metabolic syndrome.

Vimentin is necessary for colony growth of human diploid keratinocytes.

The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5ß1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5ß1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

Glucocorticoid paradoxically recruits adipose progenitors and impairs lipid homeostasis and glucose transport in mature adipocytes.

Chronic treatment with glucocorticoids increases the mass of adipose tissue and promotes metabolic syndrome. However little is known about the molecular effects of dexamethasone on adipose biology. Here, we demonstrated that dexamethasone induces progenitor cells to undergo adipogenesis. In the adipogenic pathway, at least two cell types are found: cells with the susceptibility to undergo staurosporine-induced adipose conversion and cells that require both staurosporine and dexamethasone to undergo adipogenesis. Dexamethasone increased and accelerated the expression of main adipogenic genes such as pparg2, cebpa and srebf1c. Also, dexamethasone altered the phosphorylation pattern of C/EBPß, which is an important transcription factor during adipogenesis. Dexamethasone also had effect on mature adipocytes mature adipocytes causing the downregulation of some lipogenic genes, promoted a lipolysis state, and decreased the uptake of glucose. These paradoxical effects appear to explain the complexity of the action of glucocorticoids, which involves the hyperplasia of adipose cells and insulin resistance.

EDF-1 downregulates the CaM/Cn/NFAT signaling pathway during adipogenesis.

The endothelial differentiation factor-1 (EDF-1) is a calmodulin binding protein that regulates calmodulin-dependent enzymes. In endothelial cells, this factor can form a protein complex with calmodulin. We analyzed the relationship between this factor and the members of calmodulin/calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway during adipogenesis of 3T3-F442A cells. We found that the expression of edf1 is upregulated during early adipogenesis, whereas that of calcineurin gene is lowered, suggesting that this pathway should be downregulated to allow for adipogenesis to occur. We also found that EDF-1 associates with calmodulin and calcineurin, most likely inactivating calcineurin. Our results showed that EDF-1 inactivates the calmodulin/calcineurin/NFAT pathway via sequestration of calmodulin, during early adipogenesis, and we propose a mechanism that negatively regulates the activation of calcineurin through a complex formation between EDF-1 and calmodulin. This finding raises the possibility that modulating this pathway might offer some alternatives to regulate adipose biology.

Decorin gene expression and its regulation in human keratinocytes.

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

Srebf1a is a key regulator of transcriptional control for adipogenesis.

Adipogenesis is regulated by a complex cascade of transcriptional factors, but little is known about the early events that regulate the adipogenic program. Here, we report the role of the srebf1a gene in the differentiation of fibroblastic 3T3-F442A cells. We found that expression of srebf1a depended on GSK3ß activity and that GSK3ß activity was necessary for C/EBPß phosphorylation at Thr188. Knockdown of srebf1a inhibited the adipogenic program because it blocked the expression of genes encoding PPARγ2, C/EBPα, SREBP1c and even FABP4, demonstrating that SREBP1a activation is upstream of these three essential adipogenic transcription factors. Kinetic analysis during differentiation illustrated that the order of expression of adipogenic genes was the following: cebpb, srebf1a, pparg2, cebpa, srebp1c and fabp4. Our data suggest that srebf1a acts as an essential link between the GSK3ß-C/EBPß signaling axis and the beginning of the adipogenic transcriptional cascade.

Adipogenic genes on induction and stabilization of commitment to adipose conversion.

Most late events of adipose conversion are known, but those early events that lead to cell commitment, and important aspects of its mechanism remain unknown. We recently described that, in the absence of any other adipogenic factor, 4h incubation with staurosporine promotes commitment of 3T3-F442A cells to adipogenesis. This commitment consists of two stages; a first stage of 4h induction by staurosporine, and, in the absence of this drug, a second stage of stabilization which becomes completed after 40-48h from staurosporine treatment. Here, we demonstrate that pparg2 gene is expressed early after induction stage but before commitment is stabilized, whereas cebpa is highly expressed during the last part of stabilization stage. A decrease of dlk1 expression, whose down-regulation is indispensable for adipogenesis, began to take place between 24 and 48h of St-Dex incubation started, reaching the lowest levels well into the end of stabilization stage.

Staurosporine rapidly commits 3T3-F442A cells to the formation of adipocytes by activation of GSK-3beta and mobilization of calcium.

Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3beta was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl(2), which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.

Epidermal keratinocytes do not activate peripheral T-cells: interleukin-10 as a possible regulator.

The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-gamma to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-gamma-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model.

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo.

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-beta biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.

An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells.

Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance.

Interleukin-6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture.

We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED(50) of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-alpha; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.

A simple and sensitive assay for GH activity based on 3T3-F442A cell differentiation.

We describe a fast, sensitive, specific, and simple in vitro assay for GH biological activity, based on the differentiation of 3T3-F442A cells into adipocytes. The 3T3-F442A cells were directly plated at 1.5 x 10(4)cells/cm(2) in medium with or without various concentrations of human growth hormone (hGH). After 7 days, cells were lysed with buffer containing 0.5 % (v/v) Triton X-100, and adipose conversion was quantitated by the activity of the adipogenic enzyme glycerophosphate dehydrogenase. The assay is highly sensitive and specific for GH from different species. These culture conditions have shortened the time for the cells to undergo adipose differentiation, and they might also be useful to design and test drugs or agents that modify adipocyte differentiation or lipid metabolism, or for evaluation of cytotoxic and pharmacologic effects of drugs and other compounds.