Patricia M. Watson

Norepinephrine Is Required for Leptin Effects on Gene Expression in Brown and White Adipose Tissue

Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine b-hydroxylase (Dbh 2/2 ), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 mg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh 1/2 ) compared with Dbh 2/2 mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh 1/2 mice but had no effect on UCP1 expression in Dbh 2/2 . The b3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to b-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh 1/2 but not Dbh 2/2 mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh 1/2 and Dbh 2/2 mice, illustrating that norepinephrine is not required for leptin’s effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor. (Endocrinology 140: 4772‐ 4778, 1999)

Central Leptin Regulates the UCP1 and obGenes in Brown and White Adipose Tissue via Different β-Adrenoceptor Subtypes

The three known subtypes of β-adrenoreceptors (β1-AR, β2-AR, and β3-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the β-AR subtypes in mediating leptins effects on adipocyte gene expression, mice with a targeted disruption of the β3-adrenoreceptor gene (β3-AR KO) were treated with vehicle or the β1/β2-AR selective antagonist, propranolol (20 μg/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 μg/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and β3-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in β3-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from β3-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the β3-AR is required for leptin-mediated regulation ofob mRNA expression in WAT, but is interchangeable with the β1/β2-ARs in mediating leptins effect on UCP1 mRNA expression in brown adipose tissue.

Hematopoietic stem cell origin of adipocytes

OBJECTIVE It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. MATERIALS AND METHODS Using transplantation of a single enhanced green fluorescent protein-positive (EGFP(+)) HSC and primary culture, we examined generation of adipocytes from HSCs. RESULTS Adipose tissues from clonally engrafted mice showed EGFP(+) adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of EGFP(+) adipocytes. When EGFP(+) bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. CONCLUSION Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.

Mechanisms and functional consequences of PDEF protein expression loss during prostate cancer progression

Ets is a large family of transcriptional regulators with functions in most biological processes. While the Ets family gene, prostate‐derived epithelial factor (PDEF), is expressed in epithelial tissues, PDEF protein expression has been found to be reduced or lost during cancer progression. The goal of this study was to examine the mechanism for and biologic impact of altered PDEF expression in prostate cancer.

Understanding the Role of ETS-Mediated Gene Regulation in Complex Biological Processes

Ets factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, which when perturbed contribute to tumor progression. The well-documented alterations in ETS factor expression and function during cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. The anti- and prometastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such an understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection, as well as better diagnosis and staging of disease; (2) detection of minimal residual disease recurrences and evaluation of response to therapy; (3) prevention; and (4) novel treatment strategies. Increased understanding of ETS-regulated biological pathways will directly impact these areas.

CaSm (LSm-1) Overexpression in Lung Cancer and Mesothelioma Is Required for Transformed Phenotypes

CaSm (cancer-associated Sm-like) was originally identified based on elevated expression in pancreatic cancer and in several cancer-derived cell lines. It encodes a 133-amino acid protein that contains two Sm motifs found in the common snRNP proteins and the LSm (like-Sm) family of proteins. Lung tumors and mesotheliomas express high levels of CaSm mRNA and protein compared with adjacent nontumor and normal lung tissue, measured by immunohistochemistry, qRT-PCR, and Western blot analyses. In addition, several human lung cancer- and mesothelioma-derived cell lines have elevated CaSm expression. Two cell lines, transfected with and expressing antisense CaSm RNA, demonstrate altered transformed phenotypes, reducing their ability to form colonies in soft agar and tumors in SCID mice. Furthermore, RNAi-mediated reduction of CaSm RNA and protein is associated with inhibition of cellular growth. These data support the model that elevated CaSm expression in epithelial tissue contributes to the transformed state. Cell lines expressing exogenous CaSm also exhibit transformed characteristics, including increased anchorage-independent colony formation and tumor growth. Thus, the results of loss of function and gain of function studies presented both indicate that CaSm functions as an oncogene in the promotion of cellular transformation and cancer progression.

Adrenalectomy after Weaning Restoresβ 3-Adrenergic Receptor Expression in White Adipocytes from C57BL/6J-ob/ob Mice1

The role of hypercorticism in the development of compromisedβ -adrenergic signaling in adipose tissue was assessed in ob/ob mice adrenalectomized at 4 weeks of age and studied 1 and 3 weeks thereafter. Adrenalectomy prevented the rapid increase in body weight and fat deposition between 4 and 5 weeks of age in ob/ob mice and produced a phenotype indistinguishable from that of lean mice. However, adrenalectomized ob/ob mice became intermediate between lean and ob/ob mice by 7 weeks of age. Adipocyte β3-adrenergic receptor (AR) messenger RNA levels were similar between lean and adrenalectomized ob/ob mice at both time points and were 4- to 8-fold higher than messenger RNA levels in ob/ob mice. As judged by maximal activation of adenylyl cyclase by aβ 3-AR-selective agonist, adrenalectomy also restored functional activity of the β3-AR to levels above or equivalent to those seen in lean mice at both time points. The present results suggest that development of hypercorticism at or before weaning in ob/ob mice r...

Hematopoietic stem cell origin of mesenchymal cells: opportunity for novel therapeutic approaches

There has been a general belief that there are two types of adult stem cells, i.e., hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), each with distinctly different functions. According to this dogma, HSCs produce blood cells, while MSCs are thought to generate a number of non-hematopoietic cells including fibroblasts, adipocytes, chondrocytes and bone cells. Recently, a number of studies, including those in our laboratory based on single HSC transplantation, blurred the clear distinction between HSCs and MSCs and strongly suggested an HSC origin of the adult mesenchymal tissues. This review summarizes the experimental evidence for this new paradigm and the literature pointing out the vagary in the stem cell nature of MSCs. The concept of the HSC origin of mesenchymal cells will have many immediate and long-term impacts on the therapies of diseases and injuries of the connective tissues.

Prostate-Derived ETS Factor Regulates Epithelial-to-Mesenchymal Transition through Both SLUG-Dependent and Independent Mechanisms

The 5-year survival rate is very low when breast cancer becomes metastatic. The metastatic process is governed by a network of molecules of which SLUG is known to play a major role as a regulator of epithelial-to-mesenchymal transition (EMT). Prostate-derived ETS factor (PDEF) has been proposed as a tumor suppressor, possibly through inhibition of invasion and metastasis; therefore, understanding the mechanism of PDEF regulation may help to better understand its role in breast cancer progression. This study shows for the first time that the transcription factor SLUG is a direct target of PDEF in breast cancer. We show that the expression of PDEF is able to suppress/dampen EMT through the negative regulation of SLUG. In addition, we show that PDEF is also able to regulate downstream targets of SLUG, namely E-cadherin, in both SLUG-dependent and -independent manners, suggesting a critical role for PDEF in regulating EMT.

FLI1 expression is correlated with breast cancer cellular growth, migration, and invasion and altered gene expression.

ETS factors have been shown to be dysregulated in breast cancer. ETS factors control the expression of genes involved in many biological processes, such as cellular proliferation, differentiation, and apoptosis. FLI1 is an ETS protein aberrantly expressed in retrovirus-induced hematological tumors, but limited attention has been directed towards elucidating the role of FLI1 in epithelial-derived cancers. Using data mining, we show that loss of FLI1 expression is associated with shorter survival and more aggressive phenotypes of breast cancer. Gain and loss of function cellular studies indicate the inhibitory effect of FLI1 expression on cellular growth, migration, and invasion. Using Fli1 mutant mice and both a transgenic murine breast cancer model and an orthotopic injection of syngeneic tumor cells indicates that reduced Fli1 contributes to accelerated tumor growth. Global expression analysis and RNA-Seq data from an invasive human breast cancer cell line with over expression of either FLI1 and another ETS gene, PDEF, shows changes in several cellular pathways associated with cancer, such as the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathways. This study demonstrates a novel role for FLI1 in epithelial cells. In addition, these results reveal that FLI1 down-regulation in breast cancer may promote tumor progression.