Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

Adipocyte determination-and differentiation-dependent factor 1 (ADD1), a member of the basic helix-loophelix (bHLH) family of transcription factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Using PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 has dual DNA sequence specificity, binding to both an E-box motif (ATCACGTGA) and a non-E-box sequence previously shown to be important in cholesterol metabolism, sterol regulatory element 1 (SRE-1; ATCACCCCAC). The ADD1/SREBP1 consensus E-box site is similar to a regulatory sequence designated the carbohydrate response element, defined by its ability to regulate transcription in response to carbohydrate in genes involved in fatty acid and triglyceride metabolism in liver and fat. When expressed in fibroblasts, ADD1/SREBP1 activates transcription through both the carbohydrate response E-box element and SRE-1. Substitution of an atypical tyrosine in the basic region of ADD1/SREBP1 to an arginine found in most bHLH protein causes a restriction to only E-box binding. Conversely, substitution of a tyrosine for the equivalent arginine in another bHLH protein, upstream stimulatory factor, allows this factor to acquire a dual binding specificity similar to that of ADD1/SREBP1. Promoter activation by ADD1/SREBP1 through the carbohydrate response element E box is not sensitive to the tyrosine-to-arginine mutation, while activation through SRE-1 is completely suppressed. These data illustrate that ADD1/SREBP1 has dual DNA sequence specificity controlled by a single amino acid residue; this dual specificity may provide a novel mechanism to coordinate different pathways of lipid metabolism.The basic helix-loop-helix (bHLH) transcription factors regulate gene expression by binding to specific DNA sequences. The basic domain of these proteins controls DNA binding to sites with the consensus sequence CANNTG. This consensus sequence is referred to as the E-box motif and is present in the regulatory regions of many tissue-specific genes (6,8,13,27,29). The function of the basic domain in DNA binding has been illustrated by mutations that disrupt the interaction with DNA but not oligomerization (13, 34). The Id protein, which has no basic domain, does not bind DNA but can act as a dominant suppressor of DNA binding of certain other bHLH proteins (3, 16). The HLH domain mediates homo-and hetero-oligomerization through two amphipathic ␣ helices connected by a variable loop region (1, 3, 13, 29). The various bHLH proteins can be divided into at least three groups (30). These include the broadly expressed class A proteins (E12, E47, E2-2, and daughterless) (11, 22), the tissue-specific class B proteins (MyoD, myogenin, MRF4, and achaete-scute) (9, 14, 36), and class C proteins, which feature a tandem arrangement of bHLH and leucine zipper (LZ) motifs (c-Myc, Max, upstream stimulatory factor [USF], AP4, TFE3, and TFEB) (2,5,7,10,21,24).Adipocyte determination-and differentiation-dependent factor...

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Intracellular calcium ([Ca(2+)](i)) modulates adipocyte lipid metabolism and inhibits the early stages of murine adipogenesis. Consequently, we evaluated effects of increasing [Ca(2+)](i) in early and late stages of human adipocyte differentiation. Increasing [Ca(2+)](i) with either thapsigargin or A23187 at 0-1 h of differentiation markedly suppressed differentiation, with a 40-70% decrease in triglyceride accumulation and glycerol-3 phosphate dehydrogenase (GPDH) activity (P < 0.005). However, a 1-h pulse of either agent at 47-48 h only modestly inhibited differentiation. Sustained, mild stimulation of Ca(2+) influx with either agouti protein or 10 mM KCl-induced depolarization during 0-48 h of differentiation inhibited triglyceride accumulation and GPDH activity by 20-70% (P < 0.05) and markedly suppressed peroxisome proliferator-activated receptor gamma (PPARgamma) expression. These effects were reversed by Ca(2+) channel antagonism. In contrast, Ca(2+) pulses late in differentiation (71-72 h or 48-72 h) markedly increased these markers of differentiation. Thus increasing [Ca(2+)](i) appears to exert a biphasic regulatory role in human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling.