The LIM-Only Protein Four and a Half LIM Domain Protein 2 Attenuates Development of Psoriatic Arthritis by Blocking Adam17-Mediated Tumor Necrosis Factor Release.

Four and a half LIM domain protein 2 (Fhl2) is an intracellular adaptor molecule with a high protein-protein interaction capacity. It acts as a modulator of several signaling molecules in the cytosol and as a cofactor of transcription in the nucleus. Recent studies suggest the role of Fhl2 in tissue repair and the anti-inflammatory response. Herein, we show that Fhl2-deficient mice develop a more severe psoriatic arthritis disease under induction of the inducible human tumor necrosis factor (hTNF) transgene than wild-type mice. The disease was accompanied by increased infiltration of activated macrophages and T regulatory cells in skin and digit joints as well as by increased expression of matrix metalloproteases and bone-specific proteases. The more severe pathogenesis of psoriatic arthritis in Fhl2 knockout mice coincided with enhanced levels of soluble hTNF cytokine, but surprisingly not with transcription of the hTNF transgene. Studying the shedding of cell membrane-bound hTNF by Adam17, a known Fhl2 interacting protein, revealed an enhanced release of TNF in the absence of Fhl2. In summary, our results show that Fhl2 anticipates the emerging inflammation and specifically the development of psoriatic arthritis by impeding the Adam17-mediated release of TNF.

miR-126-3p Promotes Matrix-Dependent Perivascular Cell Attachment, Migration and Intercellular Interaction.

microRNAs (miRNAs) can regulate the interplay between perivascular cells (PVC) and endothelial cells (EC) during angiogenesis, but the relevant PVC-specific miRNAs are not yet defined. Here, we identified miR-126-3p and miR-146a to be exclusively upregulated in PVC upon interaction with EC, determined their influence on the PVC phenotype and elucidate their molecular mechanisms of action. Specifically the increase of miR-126-3p strongly promoted the motility of PVC on the basement membrane-like composite and stabilized networks of EC. Subsequent miRNA target analysis showed that miR-126-3p inhibits SPRED1 and PLK2 expression, induces ERK1/2 phosphorylation and stimulates TLR3 expression to modulate cell-cell and cell-matrix contacts of PVC. Gain of expression experiments in vivo demonstrated that miR-126-3p stimulates PVC coverage of newly formed vessels and transform immature into mature, less permeable vessels. In conclusion we showed that miR-126-3p regulates matrix-dependent PVC migration and intercellular interaction to modulate vascular integrity. Stem Cells 2016;34:1297-1309.

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

AIM: Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing. METHODS: Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs. RESULTS: Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1ß and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome. CONCLUSION: RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

Macrophage-mediated psoriasis can be suppressed by regulatory T lymphocytes.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1KO or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1KO or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²âº-([Ca²âº](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²âº](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²âº signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC50(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

miR-127-3p Is an Epigenetic Activator of Myofibroblast Senescence Situated within the MicroRNA-Enriched Dlk1-Dio3‒Imprinted Domain on Mouse Chromosome 12.

During wound healing, fibroblasts differentiate into nonproliferative contractile myofibroblasts, contribute to skin repair, and eventually undergo apoptosis or become senescent. MicroRNAs are post-transcriptional regulators of gene expression networks that control cell fate and survival and may also regulate senescence. In this study, we determined the regulated microRNAs in myofibroblasts isolated from wounds and analyzed their role in senescent myofibroblast formation. Transcriptome profiling showed that a 200 kilobase pair region of the Dlk1-Dio3‒imprinted domain on mouse chromosome 12 encodes for most of the upregulated microRNAs in the entire genome of mouse myofibroblasts. Among those, miR-127-3p induced a myofibroblast-like phenotype associated with a block in proliferation. Molecular analysis revealed that miR-127-3p induced a prolonged cell cycle arrest with unique molecular features of senescence, including the activation of the senescence-associated ß-galactosidase, increase in p53 and p21 levels, inhibition of lamin B1, proliferation factors, and the production of senescence-associated inflammatory and extracellular matrix‒remodeling components. Hence, miR-127-3p emerges as an epigenetic activator regulating the transition from repair to remodeling during skin wound healing but may also induce age-related defects, pathological scarring, and fibrosis, all linked to myofibroblast senescence.

Identification of a myofibroblast-specific expression signature in skin wounds.

After skin injury fibroblasts migrate into the wound and transform into contractile, extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury, their number reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. Most of these genes were linked with the extracellular region and cell periphery including genes encoding for extracellular matrix proteins. A unique panel of core matrisome and matrisome-associated genes was differentially expressed in myofibroblasts and several genes not yet known to be linked to myofibroblast-mediated wound healing were found (e.g. Col24a1, Podnl1, Bvcan, Tinagl1, Thbs3, Adamts16, Adamts19, Cxcl's, Ccl's). In addition, a complex network of G protein-coupled signaling events was regulated in myofibroblasts (e.g. Adcy1, Plbc4, Gnas). Hence, this first characterization of a myofibroblast-specific expression profile at the peak of in situ granulation tissue formation provides important insights into novel target genes that may control excessive ECM deposition during fibrotic reactions.

PECAM1(+)/Sca1(+)/CD38(+) vascular cells transform into myofibroblast-like cells in skin wound repair.

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1(+)/Sca1(+) vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1(+)/Sca1(+)/CD38(+) cells proliferate upon wounding and could give rise to α-SMA(+) myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1(+)/Sca1(+)/CD38(+) vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1(+)/Sca1(+)/CD38(+) cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.