Biomedical subjects

Z Elizabeth Floyd

Publications and source records attributed to Z Elizabeth Floyd.

11 recordsLinked to original sources

PubMed2006

Secretome of primary cultures of human adipose-derived stem cells: modulation of serpins by adipogenesis.

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ. Adipocyte-derived "adipokines" such as adiponectin, leptin, and visceral adipose tissue-derived serine protease inhibitor (vaspin) exert hormone-like activities at the systemic level. In this study, we examined the secretome of primary cultures of human subcutaneous adipose-derived stem cells as an in vitro model of adipogenesis. Conditioned media obtained from four individual female donors after culture in uninduced or adipogenic induced conditions were compared by two-dimensional gel electrophoresis and tandem mass spectrometry. Over 80 individual protein features showing > or =2-fold relative differences were examined. Approximately 50% of the identified proteins have been described previously in the secretome of murine 3T3-L1 preadipocytes or in the interstitial fluid derived from human mammary gland adipose tissue. As reported by others, we found that the secretome included proteins such as actin and lactate dehydrogenase that do not display a leader sequence or transmembrane domain and are classified as "cytoplasmic" in origin. Moreover we detected a number of established adipokines such as adiponectin and plasminogen activator inhibitor 1. Of particular interest was the presence of multiple serine protease inhibitors (serpins). In addition to plasminogen activator inhibitor 1, these included pigment epithelium-derived factor (confirmed by Western immunoblot), placental thrombin inhibitor, pregnancy zone protein, and protease C1 inhibitor. These findings, together with the recent identification of vaspin, suggest that the serpin protein family warrants further proteomics investigation with respect to the etiology of obesity and type 2 diabetes.

3T3-L1 Cells↗

PubMed2006

Degradation of STAT5 proteins in 3T3-L1 adipocytes is induced by TNF-{alpha} and cycloheximide in a manner independent of STAT5A activation.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has been implicated as a causative factor in obesity-linked insulin resistance. It is commonly accepted that macrophage-derived TNF-alpha acts in a paracrine manner on adjacent adipocytes to inhibit the expression of various adipocyte genes and to attenuate insulin signaling. Several studies have revealed that signal transducer and activator of transcription (STAT)5 proteins are modulated during adipogenesis and can modulate the transcription of some adipocyte genes. In this study, we demonstrate that TNF-alpha treatment, in the presence of cycloheximide, also results in the rapid turnover of STAT5A and STAT5B in a process that is independent of STAT5 activation by tyrosine phosphorylation. In addition, STAT5B is more labile than STAT5A under these conditions, suggesting that the COOH terminus of STAT5 may be involved in the turnover of each protein. Initial characterization of the TNF-alpha and cycloheximide-mediated degradation of STAT5 indicates that inhibition of the proteasome stabilizes both forms of STAT5 in the presence of TNF-alpha. In addition, the use of an NF-kappaB inhibitor results in the stabilization of STAT5A in the presence of TNF-alpha and cycloheximide, indicating that the degradation of STAT5 proteins under these conditions may involve the NF-kappaB pathway. STAT5 proteins are abundantly expressed in mature adipocytes and are normally extremely stable proteins under a wide range of conditions. However, our results demonstrate that the potentiation of TNF-alpha-mediated signaling in the presence of cyclohexmide is associated with a significant increase in the degradation of STAT5 proteins in 3T3-L1 adipocytes.

3T3-L1 Cells↗

PubMed2006

Playing with bone and fat.

The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Classical in vitro and in vivo studies strongly support an inverse relationship between the commitment of bone marrow-derived mesenchymal stem cells or stromal cells to the adipocyte and osteoblast lineage pathways. In this review, we focus on the recent literature exploring the mechanisms underlying these differentiation events and discuss their implications relevant to osteoporosis and regenerative medicine.

Adipocytes↗

PubMed2006

The immunogenicity of human adipose-derived cells: temporal changes in vitro.

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase-digested human adipose tissue-derived stromal vascular fraction cells (SVFs) relative to passaged, plastic-adherent adipose-derived stem cells (ASCs). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose-derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic-associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen-DR [HLA-DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

Adipocytes↗

PubMed2006

Characterization of peripheral circadian clocks in adipose tissues.

First described in the suprachiasmatic nucleus, circadian clocks have since been found in several peripheral tissues. Although obesity has been associated with dysregulated circadian expression profiles of leptin, adiponectin, and other fat-derived cytokines, there have been no comprehensive analyses of the circadian clock machinery in adipose depots. In this study, we show robust and coordinated expression of circadian oscillator genes (Npas2, Bmal1, Per1-3, and Cry1-2) and clock-controlled downstream genes (Rev-erb alpha, Rev-erb beta, Dbp, E4bp4, Stra13, and Id2) in murine brown, inguinal, and epididymal (BAT, iWAT, and eWAT) adipose tissues. These results correlated with respective gene expression in liver and the serum markers of circadian function. Through Affymetrix microarray analysis, we identified 650 genes that shared circadian expression profiles in BAT, iWAT, and liver. Furthermore, we have demonstrated that temporally restricted feeding causes a coordinated phase-shift in circadian expression of the major oscillator genes and their downstream targets in adipose tissues. The presence of circadian oscillator genes in fat has significant metabolic implications, and their characterization may have potential therapeutic relevance with respect to the pathogenesis and treatment of diseases such as obesity, type 2 diabetes, and the metabolic syndrome.

Adiponectin↗

PubMed2005

Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.

Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32. Colony-forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell-associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell-associated marker CD34 was at peak levels in the SVF cells and/or early-passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug-resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell-associated markers (CD31, CD144 or VE-cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose-derived cells in fetal bovine serum-supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

Adipose Tissue↗

PubMed2005

Proteomic analysis of primary cultures of human adipose-derived stem cells: modulation by Adipogenesis.

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by > or = 2-fold, whereas 13 showed a > or = 3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin alpha phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.

Adipose Tissue↗

PubMed2004

STAT 5 activators can replace the requirement of FBS in the adipogenesis of 3T3-L1 cells.

The 3T3-L1 cells differentiate into fat cells that have many properties of native adipocytes including: substantial lipid accumulation, insulin sensitivity, and the ability to secrete endocrine hormones. A substantial expense in using these cells is fetal bovine serum (FBS), a critical component of efficient adipogenesis. Our recent studies on STAT 5 proteins have revealed that these transcription factors are phosphorylated and translocate to the nucleus immediately after the initiation of differentiation. Studies by several other laboratories also suggest that STAT 5 proteins can have pro-adipogenic properties. Growth hormone (GH) and prolactin (PRL) are both potent activators of STAT 5A and STAT 5B proteins. Since, FBS has high concentrations of GH; we examined the ability of GH to replace FBS as a component of the differentiation cocktail for 3T3-L1 cells. Our studies revealed that FBS was not required for the adipogenesis of 3T3-L1 cells if GH or PRL was added to the differentiation cocktail. Adipogenesis was judged by Oil Red O staining and expression of adipocyte marker genes. Hence, we have developed a substantially less expensive method for differentiating 3T3-L1 cells without FBS, thiazolidinediones, or expensive cytokines.

3T3 Cells↗

PubMed2004

Control of peroxisome proliferator-activated receptor gamma2 stability and activity by SUMOylation.

OBJECTIVE: To determine whether small ubiquitin-related modifier (SUMO)ylation of lysine 107 plays a role in regulating the activity of peroxisome proliferator-activated receptor gamma (PPARgamma). RESEARCH METHODS AND PROCEDURES: Transient expression of wild-type and K107R-PPARgamma2 in the NIH 3T3 fibroblast cell line was carried out in conjunction with half-life studies, luciferase activity assays, and indirect immunofluorescence localization studies. Additional in vitro analysis was carried out using recombinant SUMOylation pathway proteins along with in vitro transcribed and translated wild-type or K107R-PPARgamma2 to examine the SUMO-1 modification state of wild-type and SUMO-deficient K107R-PPARgamma2. RESULTS: While examining PPARgamma2 for potential ubiquitylation sites, we identified a strong consensus site for SUMO modification that contains lysine 107. In vitro, SUMOylation studies showed that lysine 107 of PPARgamma2 is a major SUMOylation site and that at least one other SUMOylation site is present in PPARgamma. In addition, our results demonstrated that SUMO-1 affects PPARgamma stability and transcriptional activity but not the nuclear localization of PPARgamma. DISCUSSION: These results indicated that SUMOylation plays a role in regulating PPARgamma, both indirectly and directly by modification of lysine 107. Because PPARgamma is regulated in numerous animal models of obesity, understanding the covalent modifications of PPARgamma may enhance our understanding of the metabolic syndrome.

Adipose Tissue↗

PubMed2003

STAT5A promotes adipogenesis in nonprecursor cells and associates with the glucocorticoid receptor during adipocyte differentiation.

The differentiation of adipocytes is regulated by the activity of a variety of transcription factors, including peroxidase proliferator-activated receptor (PPAR)-gamma and C/EBPalpha. Our current study demonstrates that ectopic expression of STAT5A, such as that of PPAR-gamma and C/EBPalpha, promotes adipogenesis in two nonprecursor fibroblast cell lines. Using morphologic and biochemical criteria, we have demonstrated that STAT5A and the combination of STAT5A and STAT5B are sufficient to induce the expression of early and late adipogenic markers in BALB/c and NIH-3T3 cells. Yet, the ectopic expression of STAT5B alone does not induce the expression of adipocyte genes, but enhances the induction of these genes in cells also expressing STAT5A. This finding suggests that STAT5A and STAT5B do not function identically in adipocytes. In addition, these studies demonstrate that the phosphorylation of STAT5 proteins may play a role in adipogenesis. Moreover, we have shown that STAT5A is associated with the glucocorticoid receptor during adipogenesis in a highly regulated manner.

3T3 Cells↗

PubMed2001

Interferon-gamma-mediated activation and ubiquitin-proteasome-dependent degradation of PPARgamma in adipocytes.

Interferon-gamma (IFNgamma) treatment of adipocytes results in a down-regulation of the peroxisome proliferator-activated receptor gamma (PPARgamma). The decrease in PPARgamma expression is mediated by inhibition of PPARgamma synthesis and increased degradation of PPARgamma. In this study, we demonstrate that both PPARgamma1 and PPARgamma2 are targeted to the proteasome under basal conditions and that PPARgamma1 is more labile than PPARgamma2. The IFNgamma-induced increase in PPARgamma turnover is blocked by proteasome inhibition and is accompanied by an increase in PPARgamma-polyubiquitin conjugates. In addition, IFNgamma treatment results in the transcriptional activation of PPARgamma. Similar to ligand-dependent activation of PPARgamma, IFNgamma-induced activation was greater in the phosphorylation-deficient S112A form of PPARgamma when compared with wild-type PPARgamma. Moreover, the inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an inhibition in the decay of PPARgamma proteins, indicating that serine phosphorylation influences the degradation of PPARgamma in fat cells. Our results also demonstrate that the proteasome-dependent degradation of PPARgamma does not require nuclear export. Taken together, these results indicate that PPARgamma is targeted to the ubiquitin-proteasome pathway for degradation under basal conditions and that IFNgamma leads to an increased targeting of PPARgamma to the ubiquitin-proteasome system in a process that is affected by ERK-regulated serine phosphorylation of PPARgamma proteins.

3T3 Cells↗