Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor–dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF+ inflammatory macrophages; however, in some cases, medication redirected them into a state that is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions.

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase old-35 ), a type I IFN-inducible 3′-5′ exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase old-35 identified miRNAs differentially and specifically regulated by hPNPase old-35 . One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27 kip1 , displayed robust down-regulation with ensuing up-regulation of p27 kip1 by expression of hPNPase old-35 , which also occurred in multiple human melanoma cells upon IFN-β treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase old-35 . Inhibition of hPNPase old-35 by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-β-mediated growth inhibition, accentuating the importance of hPNPase old-35 induction and miR-221 down-regulation in mediating IFN-β action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase old-35 might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.M icroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs that regulate gene expression at the posttranscriptional level and play important roles in a multiplicity of biological functions, including cell differentiation, tumorigenesis, apoptosis, and metabolism (1). miRNA genes are initially transcribed principally by either RNA polymerase II or RNA polymerase III as long primary transcripts, which are further processed by the nuclear RNase Drosha and cytoplasmic RNase Dicer to produce precursor miRNAs and mature miRNAs, respectively (2). miRNAs recognize and bind to partially complementary sites in the 3′ UTRs of target mRNAs, resulting in either translational repression or target degradation (3). The steadystate levels of miRNAs, crucial for its profound impact on a wide array of biological processes (4, 5), are presumably determined by the opposing activities of miRNA biogenesis and degradation. Although the framework of miRNA biogenesis is established, factors involved in miRNA dysregulation remain unknown. Recent work from Ramachandran and Chen (6) documented that an exoribonuclease encoded by small rna degrading nuclease (sdn) gene degrades mature miRNAs in Arabidopsis. Although in human cells the posttranscriptional control of miRNA is poorly defined, it can be hypothesized that enzymes involved in miRNA metabolism evolved from enzymes that process structural and/or catalytic RNAs, a view supported by the fact that a number of known molecules involved...

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored an hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested crosstalk between these two proteins. Further, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Lastly, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.