Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma, encodes 25 mature viral miRNAs. MCP-1-induced protein-1 (MCPIP1), a critical regulator of immune homeostasis, has been shown to suppress miRNA biosynthesis via cleavage of precursor miRNAs through its RNase domain. We demonstrate that MCPIP1 can directly cleave KSHV and EBV precursor miRNAs and that MCPIP1 expression is repressed following de novo KSHV infection. In addition, repression with siRNAs to MCPIP1 in KSHV-infected cells increased IL-6 and KSHV miRNA expression, supporting a role for MCPIP1 in IL-6 and KSHV miRNA regulation. We also provide evidence that KSHV miRNAs repress MCPIP1 expression by targeting the 3'UTR of MCPIP1. Conversely, expression of essential miRNA biogenesis components Dicer and TRBP is increased following latent KSHV infection. We propose that KSHV infection inhibits a negative regulator of miRNA biogenesis (MCPIP1) and up-regulates critical miRNA processing components to evade host mechanisms that inhibit expression of viral miRNAs. KSHV-mediated alterations in miRNA biogenesis represent a novel mechanism by which KSHV interacts with its host and a new mechanism for the regulation of viral miRNA expression.

The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1(-/-)) or depletion (Nrf2(-/-)) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

Studies from a number of laboratories suggest that modulation of cytokine expression plays an integral role in the immunomodulatory activity of opioids. Previously, our laboratory reported that activation of the mu-opioid receptor induced the expression of CCL2, CCL5, and CXCL10, as well as CCR5 and CXCR4. Previous work has also suggested the possibility that TGF-beta may participate in the opioid-induced regulation of immune competence, and in the present study, we set out to determine the role of this cytokine in the control of chemokine and chemokine receptor expression. We found that D-ala(2),N-Me-Phe(4)-Gly-ol(5)enkephalin (DAMGO), a highly selective mu-opioid agonist, induced the expression of TGF-beta1 expression at the protein and mRNA levels. In turn, the addition of TGF-beta1 was found to induce CCL5 and CXCR4 expression but not CCL2, CXCL10, or CCR5. Further analysis showed that pretreatment with neutralizing anti-TGF-beta1 blocked the ability of DAMGO to induce CCL5 or CXCR4. Similarly, pretreatment with cycloheximide prevented CCL5 or CXCR4 mRNA expression, consistent with the observation that DAMGO induction of chemokine and chemokine receptor expression requires newly synthesized TGF-beta1 protein. These results describe a common molecular basis for the activation of chemokine and chemokine receptor expression and may permit the development of strategies to inhibit certain undesirable immunological properties of micro-opioid agonists such as morphine and heroin.