UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which we show here are competitive pathways. STAU1-and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with our finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. We discuss the remarkable balance between SMD and the two types of NMD in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis.[Keywords: Staufen1-mediated mRNA decay; nonsense-mediated mRNA decay; Staufen1; UPF proteins; premature termination codon; myogenesis] Supplemental material is available at http://www.genesdev.org.

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G 2 /M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.

Objective Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. Methods In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and age and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of pro-inflammatory/pain-associated interleukin 6 (IL-6) and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. Results Dectin-1, a surface receptor that binds C. albicans cell wall glucan, was significantly elevated in vestibular versus external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C. albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C. albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of NFκB (pro-inflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C. albicans, in keeping with observations that Dectin-1 signals through the NFκB pathway. Conclusion These findings implicate that a fibroblast-mediated pro-inflammatory response to C. albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.

Fibroblast strains were derived from two regions of the lower genital tract of localized provoked vulvodynia (LPV) cases and pain-free controls. Sixteen strains were derived from four cases and four controls, age and race matched, following pre-sampling mechanical pain threshold assessments. Strains were challenged with six separate stimuli: live yeast species (C. albicans, C. glabrata, C. tropicalis, and S. cerevisiae), yeast extract (zymosan), or inactive vehicle. Production of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were pro-inflammatory response measures. Highest IL-6 and PGE2 occurred with vestibular strains following C. albicans, C. glabrata, and zymosan challenges, resulting in the ability to significantly predict IL-6 and PGE2 production by genital tract location. Following C. albicans and C. glabrata challenge of all sixteen fibroblast strains, adjusting for dual sampling of subjects, PGE2 and IL-6 production significantly predicted the pre-sampling pain threshold from the genital tract site of sampling. At the same location of pain assessment and fibroblast sampling, in situ immunohistochemical (IHC)(+) fibroblasts for IL-6 and Cox-2 were quantified microscopically. The correlation between IL-6 production and IL-6 IHC(+) was statistically significant yet biological significance is unknown because of the small number of IHC(+) IL-6 fibroblasts identified. A low fibroblast IL-6 IHC(+) count may result from most IL-6 produced by fibroblasts existing in a secreted, extracellular state. Enhanced, site-specific, innate immune responsiveness to yeast pathogens by fibroblasts may be an early step in LPV pathogenesis. Fibroblast strain testing may offer an attractive/objective marker of LPV pathology in women with vulvodynia of inflammatory origin.

Corneal scarring, whether caused by trauma, laser refractive surgery, or infection, remains a significant problem for humans. Certain ligands for peroxisome proliferator-activated receptor gamma (PPARγ) have shown promise as antiscarring agents in a variety of body tissues. In the cornea, their relative effectiveness and mechanisms of action are still poorly understood. Here, we contrasted the antifibrotic effects of three different PPARγ ligands (15-deoxy-Δ12,14-prostaglandin J2, troglitazone, and rosiglitazone) in cat corneal fibroblasts. Western blot analyses revealed that all three compounds reduced transforming growth factor (TGF)-β1-driven myofibroblast differentiation and up-regulation of α-smooth muscle actin, type I collagen, and fibronectin expression. Because these effects were independent of PPARγ, we ascertained whether they occurred by altering phosphorylation of Smads 2/3, p38 mitogen-activated protein kinase, stress-activated protein kinase, protein kinase B, extracellular signal-regulated kinase, and/or myosin light chain 2. Only p38 mitogen-activated protein kinase phosphorylation was significantly inhibited by all three PPARγ ligands. Finally, we tested the antifibrotic potential of troglitazone in a cat model of photorefractive keratectomy-induced corneal injury. Topical application of troglitazone significantly reduced α-smooth muscle actin expression and haze in the stromal ablation zone. Thus, the PPARγ ligands tested here showed great promise as antifibrotics, both in vitro and in vivo. Our results also provided new evidence for the signaling pathways that may underlie these antifibrotic actions in corneal fibroblasts.

Worldwide obesity rates are at epidemic levels, and new insight into the regulation of obesity and adipogenesis are required. Thy1 (CD90), a cell surface protein with an enigmatic function, is expressed on subsets of fibroblasts and stem cells. We used a diet-induced obesity model to show that Thy1-null mice gain weight at a faster rate and gain 30% more weight than control C57BL/6 mice. During adipogenesis, Thy1 expression is lost in mouse 3T3-L1 cells. Overexpression of Thy1 blocked adipocyte formation and reduced mRNA and protein expression of an adipocyte marker, fatty acid-binding protein 4, by 5-fold. Although preadipocyte fibroblasts expressed Thy1 mRNA and protein, adipocytes from mouse and human fat tissue had almost undetectable Thy1 levels. Thy1 decreases the activity of the adipogenic transcription factor PPARγ by more than 60% as shown by PPARγ-dependent reporter assays. Using both genetic and pharmacologic approaches, we show Thy1 expression dampens PPARγ by inhibiting the activity of the Src-family kinase, Fyn. Thus, these studies reveal Thy1 blocks adipogenesis and PPARγ by inhibiting Fyn and support the idea that Thy1 is a novel therapeutic target in obesity.

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.