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The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPdelta in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPbeta, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBPdelta was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPbeta(-/-) mice, and C/EBPbeta was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPbeta-deficient animals, especially males. Thus, C/EBPbeta contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.

This laboratory module simulates the process used by working scientists to ask and answer a question of biological interest. Instructors facilitate acquisition of knowledge using a comprehensive, inquiry-based approach in which students learn theory, hypothesis development, experimental design, and data interpretation and presentation. Using inflammation in macrophages as a model system, students perform a series of molecular biology techniques to address the biological question: “Does stimulus ‘X’ induce inflammation?” To ask this question, macrophage cells are treated with putative inflammatory mediators and then assayed for evidence of inflammatory response. Students become familiar with their assigned mediator and the relationship between their mediator and inflammation by conducting literature searches, then using this information to generate hypotheses which address the effect of their mediator on induction of inflammation. The cellular and molecular approaches used to test their hypotheses include transfection and luciferase reporter assay, immunoblot, fluorescence microscopy, enzyme-linked immunosorbent assay, and quantitative PCR. Quantitative and qualitative reasoning skills are developed through data analysis and demonstrated by successful completion of post-lab worksheets and the generation and oral presentation of a scientific poster. Learning objective assessment relies on four instruments: pre-lab quizzes, post-lab worksheets, poster presentation, and posttest. Within three cohorts (n = 85) more than 95% of our students successfully achieved the learning objectives.

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative C T method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events. It requires only 4-5 hr of dedicated laboratory time and focuses on use of the primary literature, data analysis, and interpretation. Importantly, this exercise provides a mechanism to introduce the concept of differential gene expression and provides a starting point for development of more complex guided or open inquiry projects for students moving into upper level molecular biology, immunology, and biochemistry course work. V C 2015 by the International Union of Biochemistry and Molecular Biology, 43(4): 273-282, 2015.

The inflammatory response represents a significant component of brain related disease responsible for neuronal degeneration. However, in some cases, inflammation is also a necessary first step during innate immune responses involved in mediating tissue repair. HMGB‐1 is a nuclear, non‐histone, DNA binding protein that is normally involved in chromatin stabilization and transcriptional regulation. It also has a well established role in mediating pro‐inflammatory events following passive release from necrotic neurons and glia. Similarly, C/EBPβ and C/EBPδ are nuclear transcription factors known to promote the expression of pro‐inflammatory genes following ischemic insult; and, both C/EBP β and C/EBP δ have been shown to mediate tissue repair following inflammatory stimuli in various cell types. Here, we report the results of our investigations into defining the role of C/EBP proteins in mediating the downstream signaling events that promote inflammation in response to passive HMGB‐1 release. Primary neurons and glia, subjected to hypoxia, were examined by Western analysis to determine the levels of C/EBPβ and C/EBPδ. In response to hypoxia, both C/EBP proteins were observed to be upregulated. In addition, cells treated with purified HMGB1 protein also showed an increase in C/EBP expression. Taken together, these data suggest that C/EBP proteins may function as downstream targets of HMGB1 signaling to promote HMGB1 mediated cellular responses to tissue injury.