The GIY-YIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis.

Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1Δ/Δ-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception.

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

Genetically corrected iPSCs as cell therapy for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen, resulting in fragile skin and mucous membranes that blister easily in response to mechanical stress. Induced pluripotent stem cells (iPSCs) carry the potential to fundamentally change cell-based therapies for human diseases, in particular for RDEB, for which no effective treatments are available. To provide proof of principle on the applicability of iPSCs for the treatment of RDEB, we developed iPSCs from type VII collagen (Col7a1) mutant mice that exhibited skin fragility and blistering resembling human RDEB. Genetically repaired iPSCs could be differentiated into functional fibroblasts that reexpressed and secreted type VII collagen. Corrected iPSC-derived fibroblasts did not form tumors in vivo and could be traced up to 16 weeks after intradermal injection. Moreover, iPSC-based cell therapy resulted in faithful and long-term restoration of type VII collagen deposition at the epidermal-dermal junction of Col7a1 mutant mice. Intradermal injection of genetically repaired iPSC-derived fibroblasts restored the mechanical resistance to skin blistering in mice with RDEB, suggesting that RDEB skin could be effectively and safely repaired using iPSC-based cell therapy.

Construction of a global pain systems network highlights phospholipid signaling as a regulator of heat nociception.

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression.

Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRas(G12D)-driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Epidermal loss of JunB leads to a SLE phenotype due to hyper IL-6 signaling.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting various tissues. Involvement of B and T cells as well as increased cytokine levels have been associated with disease manifestation. Recently, we demonstrated that mice with epidermal loss of JunB (JunB(Deltaep)) develop a myeloproliferative syndrome (MPS) due to high levels of G-CSF which are secreted by JunB-deficient keratinocytes. In addition, we show that JunB(Deltaep) mice develop a SLE phenotype linked to increased epidermal interleukin 6 (IL-6) secretion. Intercrosses with IL-6-deficient mice could rescue the SLE phenotype. Furthermore, we show that JunB binds to the IL-6 promoter and transcriptionally suppresses IL-6. Facial skin biopsies of human SLE patients similarly revealed low JunB protein expression and high IL-6, activated Stat3, Socs-1, and Socs-3 levels within lupus lesions. Thus, keratinocyte-induced IL-6 secretion can cause SLE and systemic autoimmunity. Our results support trials to use alpha-IL-6 receptor antibody therapy for treatment of SLE.

Epidermal JunB represses G-CSF transcription and affects haematopoiesis and bone formation.

Mice that lack JunB in epidermal cells are born with normal skin; however, keratinocytes hyperproliferate in vitro and on TPA treatment in vivo. Loss of JunB expression in the epidermis of adult mice affects the skin, the proliferation of haematopoietic cells and bone formation. G-CSF is a direct transcriptional target of JunB and mutant epidermis releases large amounts of G-CSF that reach high systemic levels and cause skin ulcerations, myeloproliferative disease and low bone mass. The absence of G-CSF significantly improves hyperkeratosis and prevents the development of myeloproliferative disease, but does not affect bone loss. This study describes a mechanism by which the absence of JunB in epithelial cells causes multi-organ disease, suggesting that the epidermis can act as an endocrine-like organ.

Impaired heart contractility in Apelin gene-deficient mice associated with aging and pressure overload.

Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene-targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload-induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin(-/y) and Apelin(+/y) mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.

S-nitrosylation of microtubule-associated protein 1B mediates nitric-oxide-induced axon retraction.

Treatment of cultured vertebrate neurons with nitric oxide leads to growth-cone collapse, axon retraction and the reconfiguration of axonal microtubules. We show that the light chain of microtubule-associated protein (MAP) 1B is a substrate for S-nitrosylation in vivo, in cultured cells and in vitro. S-nitrosylation occurs at Cys 2457 in the COOH terminus. Nitrosylation of MAP1B leads to enhanced interaction with microtubules and correlates with the inhibition of neuroblastoma cell differentiation. We further show, in dorsal root ganglion neurons, that MAP1B is necessary for neuronal nitric oxide synthase control of growth-cone size, growth-cone collapse and axon retraction. These results reveal an S-nitrosylation-dependent signal-transduction pathway that is involved in regulation of the axonal cytoskeleton and identify MAP1B as a major component of this pathway. We propose that MAP1B acts by inhibiting a microtubule- and dynein-based mechanism that normally prevents axon retraction.

JunD regulates lymphocyte proliferation and T helper cell cytokine expression.

Transgenic mice broadly expressing JunD (Ubi-junD(m)) appear phenotypically normal, but have strongly reduced numbers of peripheral lymphocytes. JunD overexpression in lymphocytes does not protect from numerous apoptotic insults; however, transgenic T cells proliferate poorly and exhibit impaired activation due to reduced levels of IL-4, CD25 and CD69. Consistently, in the absence of JunD (junD(-/-)) T cells hyperproliferate following mitogen induction. Moreover, transgenic T helper (Th) 2 cells have decreased IL-4 and IL-10 expression, whereas junD(-/-) Th2 cells secrete higher amounts of both Th2 cytokines. Th1-polarized junD(-/-) CD4(+) T cells display enhanced IFN-gamma cytokine production associated with upregulated T-bet expression and downregulated expression of suppressor of cytokine signaling-1. These novel findings demonstrate a regulatory role of JunD in T lymphocyte proliferation and Th cell differentiation.