Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.

In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6-desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219-231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long-chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.

SUMMARYThe Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2 + cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2+ cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound reepithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9 + and Tcf4 + cells in the HFs closely adjacent to the wound in Lhx2 +/-mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2 +/-mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.

Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.

High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.

SummaryBone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumoursuppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

Clostridium sordellii was isolated from 37 sheep, in 29 flocks, which died suddenly between 1993 and 1995. The sheep were of all ages, but the most severe gross lesions affected lambs four to 10 weeks of age. In older weaned lambs and ewes the gross changes were less marked and more variable. Thirty sheep suffering from a variety of other conditions were examined and C sordellii was not isolated. The isolation of C sordellii has been reported only twice before from sheep in Britain, and on both occasions no detailed investigations were described. The evidence from this study indicates that C sordellii should be considered when investigating the cause of sudden death in sheep of all ages in Britain.

Bone morphogenetic protein (BMP) signalling plays a key role in the control of skin development and postnatal remodelling by regulating keratinocyte proliferation, differentiation and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and qRT-PCR analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a, in the epidermis of K14-caSmad1 mice versus wild-type controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared to wild-type controls. Finally, siRNA-mediated silencing of Bmpr-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17 and Myo5a compared to controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.