Dwight J. Klemm

CREB activation induces adipogenesis in 3T3-L1 cells.

ABSTRACT Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.

Rosiglitazone promotes development of a novel adipocyte population from bone marrow-derived circulating progenitor cells.

Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP(+) multilocular (ML) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These ML adipocytes expressed adiponectin, perilipin, fatty acid-binding protein (FABP), leptin, C/EBPalpha, and PPARgamma but not uncoupling protein-1 (UCP-1), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP(+) ML adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into ML adipocytes.

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.

Reprogramming somatic cells into an ESC‐like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage‐specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS‐derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage‐specific genes. Isolated implants of iPS cell‐derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease. STEM CELLS 2011;29:206–216

De novo generation of white adipocytes from the myeloid lineage via mesenchymal intermediates is age, adipose depot, and gender specific

It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.

CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Leprdb/db Mice

CCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPβ in hepatic lipogenesis remains undefined. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x Leprdb/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPβ deletion in Leprdb/db mice down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) and stearoyl-CoA desaturase-1 and up-regulated PPARα independent of SREBP1c. Conversely, C/EBPβ overexpression in wild-type mice increased PPARγ2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPβ or C/EBPβ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARγ2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPβ expression in hepatocytes, whereas fatty acids up-regulate C/EBPβ expression. These data provide novel evidence linking C/EBPβ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.

Depletion of cAMP-response Element-binding Protein/ATF1 Inhibits Adipogenic Conversion of 3T3-L1 Cells Ectopically Expressing CCAAT/Enhancer-binding Protein (C/EBP) α, C/EBP β, or PPARγ2

The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the “master adipogenic regulators,” CCAAT/enhancer-binding protein (C/EBP) β, peroxisome proliferator-activated receptor γ (PPARγ), and C/EBP α. In addition, activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion and prevent apoptosis of mature adipocytes. In this report we used small interfering RNAto deplete CREB and the closely related factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARγ, C/EBP α, and adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically expressing C/EBP α, C/EBP β, or PPARγ2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing both PPARγ2 and C/EBP α, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively active CREB overcame the blockade of adipogenesis due to depletion of C/EBP β but not due to loss of PPARγ2 or C/EBP α. Depletion of CREB/ATF1 did not suppress the expression of C/EBP β as we had previously observed using dominant negative forms of CREB. Finally results are presented showing that CREB promotes PPARγ2 gene transcription. The results indicate that CREB and ATF1 play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also at later stages.

Insulin Stimulates cAMP-response Element Binding Protein Activity in HepG2 and 3T3-L1 Cell Lines

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf → MEK pathway and has an impact on physiologically relevant genes in these cells.

The Pathology of Bleomycin-Induced Fibrosis Is Associated with Loss of Resident Lung Mesenchymal Stem Cells That Regulate Effector T-cell Proliferation†‡§

Tissue‐resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T‐cell response. Bleomycin‐induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin‐associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T‐cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury. STEM Cells 2011;29:725–735

Leukemic Stem Cells Evade Chemotherapy by Metabolic Adaptation to an Adipose Tissue Niche

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

CREB Downregulation in Vascular Disease. A Common Response to Cardiovascular Risk

Objective—To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence. Methods and Results—VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice. There was significant loss of CREB protein in all models. In vitro, primary culture rat aortic VSMC exposed to LDL and oxidized LDL exhibited a rapid, transient increase in CREB phosphorylation and transient phosphorylation/activation of Akt, ERK, JNK, ans p38 MAPK. Exposure to oxidized LDL, but not to LDL, for 24 to 48 hours decreased CREB protein in a dose-dependent fashion and led to nuclear exclusion of CREB. Pharmacological reactive oxygen species scavengers and inhibition of ERK activation blocked oxidized LDL-mediated CREB downregulation. Conclusion—These data support a model wherein loss of VSMC CREB protein, which renders these cells more susceptible to activation and apoptosis, is a common pathological response to vascular injury and potentially contributes to plaque progression.