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.

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.

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.

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.

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.

The transient expression of Klf4 and Klf5 during adipogenesis depends on GSK3β 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 GSK3β activity was required for Klf4 and Klf5 expression during 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.

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

Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3β-C/EBPβ-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.

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(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network.

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.

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.

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 GSK3β activity and that GSK3β activity was necessary for C/EBPβ phosphorylation at Thr188.

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.

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.

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.

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.

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.

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.

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.

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).

Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24-36 h after adipogenic stimulation: TNF-alpha inhibits commitment.

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions.

Frozen cultured sheets of epidermal keratinocytes in reepithelialization and repair of the cornea after photorefractive keratectomy.

Purpose: To determine whether frozen cultured sheets of human allogeneic epidermal keratinocytes (CEAK) improved wound repair after experimental corneal ablation by photorefractive keratectomy (PRK).

Setting: Hospital "Luis Sanchez Bulnes" de la Asociación para Evitar la Ceguera en Mexico, I.A.

Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal keratinocytes.

In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-alpha (TGF-alpha); transforming growth factor-beta (TGF-beta) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures.

Controlled clinical study of deep partial-thickness burns treated with frozen cultured human allogeneic epidermal sheets.

Numerous studies, many uncontrolled, have suggested that the application of freshly prepared human allogeneic epidermal cultures promotes faster re-epithelialization of skin donor sites and deep partial-thickness burns. We describe the results of a study of deep partial-thickness burns treated with such cultures preserved in the frozen state. The study was controlled, side-by-side comparative, and randomized.

Frozen human epidermal allogeneic cultures promote rapid healing of facial dermabrasion wounds.

Background: Clinical studies have shown that cultured human epidermal allogenic sheets promote faster reepithelization of skin donor sites and deep partial-thickness wounds.

Objective: We describe the results of a controlled, clinical study of facial dermabrasion sites treated with a single application of frozen cultured human allogenic epidermal sheets that were thawed for 5-10 minutes at room temperature before application.

Methods: Ten patients with scars from acne or of other etiology underwent facial dermabrasion.