TAM Kinases Promote Necroptosis by Regulating Oligomerization of MLKL.

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

Receptor tyrosine kinases Tyro3, Axl, and Mertk differentially contribute to antibody-induced arthritis.

Tyro3, Axl, and Mertk (abbreviated TAMs) comprise a family of homologous type 1 receptor tyrosine kinases (RTKs) that have been implicated as inhibitory receptors that dampen inflammation, but their roles in the pathogenesis of rheumatoid arthritis remains understudied. Here, to investigate TAMs in an inflammatory arthritis model, antibody-induced arthritis in single TAM-deficient mice (Tyro3- KO, Axl-KO, Mertk-KO) was induced by K/BxN serum injection. Subsequently, joint inflammation and cytokine levels, as well as the expression of Fcγ Rs and complement receptors were assessed in WT and TAM-deficient mice. Compared with littermate control mice, Axl-/- and Mertk-/- mice developed more severe antibody-induced arthritis, while in contrast, Tyro3-/- mice showed diminished joint inflammation. Concomitantly, the levels of cytokines in joints of Axl-/- and Mertk-/- mice were also significantly increased, while cytokines in the Tyro3-/- joint tissues were decreased. At the molecular and cellular level, TAMs showed distinct expression patterns, whereby monocytes expressed Axl and Mertk, but no Tyro3, while neutrophils expressed Axl and Tyro3 but little Mertk. Moreover, expression of Fcγ receptors and C5aR showed different patterns with TAMs expression, whereby FcγRIV was higher in monocytes of Axl-/- and Mertk-/- mice compared to wild-type mice, while Tyro3-/- neutrophils showed lower expression levels of FcγRI, FcγRIII and FcγRIV. Finally, expression of C5aR was increased in Mertk-/- monocytes, and was decreased in Tyro3-/- neutrophils. These data indicate that Axl, Mertk and Tyro3 have distinct functions in antibody-induced arthritis, due in part to the differential regulation of cytokines production, as well as expression of FcγRs and C5aR. Video Abstract.

Yap1 is required for maintenance of adult RPE differentiation.

Nuclear YAP1 plays a critical role in regulation of stem cell proliferation, tissue regeneration, and organ size in many types of epithelia. Due to rapid turnover of most epithelial cell types, the cytoplasmic function of YAP1 in epithelial cells has not been well studied. The retinal pigment epithelium (RPE) is a highly polarized epithelial cell type maintained at a senescence state, and offers an ideal cell model to study the active role of YAP1 in maintenance of the adult epithelial phenotype. Here, we show that the cytoplasmic function of YAP1 is essential to maintain adult RPE differentiation. Knockout of Yap1 in the adult mouse RPE caused cell depolarization and tight junction breakdown, and led to inhibition of RPE65 expression, diminishment of RPE pigments, and retraction of microvilli and basal infoldings. These changes in RPE further prompted the loss of adjacent photoreceptor outer segments and photoreceptor death, which eventually led to decline of visual function in older mice between 6 and 12 months of age. Furthermore, nuclear ß-catenin and its activity were significantly increased in mutant RPE. These results suggest that YAP1 plays an important role in active inhibition of Wnt/ß-catenin signaling, and is essential for downregulation of ß-catenin nuclear activity and prevention of dedifferentiation of adult RPE.

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation.

BACKGROUND: Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood. METHODS: Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo. RESULTS: Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl-/- and Tyro3-/- mice, but not in Mertk-/- mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl-/- and Tyro3-/- platelets, but not in Mertk-/- platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation. CONCLUSIONS: These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

Expression of cytokines in aqueous humor from fungal keratitis patients.

BACKGROUND: Although a series of reports on corneal fungal infection have been published, studies on pathogenic mechanisms and inflammation-associated cytokines remain limited. In this study, aqueous humor samples from fungal keratitis patients were collected to examine cytokine patterns and cellular profile for the pathogenesis of fungal keratitis. METHODS: The aqueous humor samples were collected from ten patients with advanced stage fungal keratitis. Eight aqueous humor samples from patients with keratoconus or corneal dystrophy were taken as control. Approximately 100 µl to 300 µl of aqueous humor in each case were obtained for examination. The aqueous humor samples were centrifuged and the cells were stained and examined under optical microscope. Bacterial and fungal cultures were performed on the aqueous humor and corneal buttons of all patients. Cytokines related to inflammation including IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were examined using multiplex bead-based Luminex liquid protein array systems. RESULTS: Fungus infection was confirmed in these ten patients by smear stains and/or fungal cultures. Bacterial and fungal cultures revealed negative results in all aqueous humor specimens. Polymorphonuclear leukocytes were the predominant infiltrating cells in the aqueous humor of fungal keratitis. At the advanced stages of fungal keratitis, the levels of IL-1ß, IL-6, IL-8, and IFN-γ in the aqueous humor were significantly increased when compared with control (p<0.01). The levels of IL-10 and TNF-α also showed an ascending trend but with no statistical significance. CONCLUSIONS: High concentration of IL-1ß, IL-6, IL-8, and IFN-γ in the aqueous humor was associated with fungal keratitis.

TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1ß, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

TAM receptor deficiency affects adult hippocampal neurogenesis.

The Tyro3, Axl and Mertk (TAM) subfamily of receptor protein tyrosine kinases functions in cell growth, differentiation, survival, and most recently found, in the regulation of immune responses and phagocytosis. All three receptors and their ligands, Gas6 (growth arrest-specific gene 6) and protein S, are expressed in the central nervous system (CNS). TAM receptors play pivotal roles in adult hippocampal neurogenesis. Loss of these receptors causes a comprised neurogenesis in the dentate gyrus of adult hippocampus. TAM receptors have a negative regulatory effect on microglia and peripheral antigen-presenting cells, and play a critical role in preventing overproduction of pro-inflammatory cytokines detrimental to the proliferation, differentiation, and survival of adult neuronal stem cells (NSCs). Besides, these receptors also play an intrinsic trophic function in supporting NSC survival, proliferation, and differentiation into immature neurons. All these events collectively ensure a sustained neurogenesis in adult hippocampus.

Mertk deficiency alters expression of micrornas in the retinal pigment epithelium cells.

Phagocytic clearance of the spent photoreceptor outer segments (OS) by RPE cells is regulated by circadian rhythm cycle and is essential for photoreceptor integrity and function. Mertk regulates RPE phagocytosis and a deficiency in Mertk causes photoreceptor degeneration and visual loss. This study aimed to investigate Mertk regulation of the microRNAs (miRNA), potentially regulating expression of their target genes, which affect phagocytosis. The differentially expressed miRNAs were identified using miRCURY(TM) microRNA Arrays from total RNA isolated at 0900 h and 1900 h from the mechanically dissociated RPE sheets of the WT and Mertk (-/-) mice, which were housed in a 12-h light-dark cycle with the lighting onset at 0700 h (7:00am). Validation of the differentially expressed miRNAs and assessment of the putative miRNA target gene expression were performed by real-time PCR. Among the differentially expressed miRNAs in the Mertk (-/-) RPE, seven miRNAs were up-regulated and 13 were down-regulated in the morning groups. Similarly, 24 miRNAs were found to be up-regulated and 13 were down-regulated in the evening groups. To search for those that may participate in regulating expression of cytoskeletal proteins, we examined the predicted target genes that might participate in phagocytosis were examined by real-time PCR. Of nine potential altered targets, four deregulated genes were myosin subunits. Notably, multiple members of the 21 up-regulated miRNAs can theoretically recognize these down-regulated mRNAs, particularly MyH14 and Myl3. This study shows that loss of Mertk alters miRNA expression, which in turn affects expression of the downstream target genes, potentially affecting phagocytosis.

Deciphering TGF-ß3 function in medial edge epithelium specification and fusion during mouse secondary palate development.

BACKGROUND: Transforming growth factor-ß3 (TGF-ß3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-ß3 functions during secondary palate fusion are still poorly understood. RESULTS: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf-ß3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-ß3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-ß3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-ß3 null mutant palatal shelves. CONCLUSIONS: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-ß3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-ß3 downstream target genes in palate medial edge epithelium differentiation.

Exosomes from retinal astrocytes contain antiangiogenic components that inhibit laser-induced choroidal neovascularization.

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.

Repression of Zeb1 and hypoxia cause sequential mesenchymal-to-epithelial transition and induction of aid, Oct4, and Dnmt1, leading to immortalization and multipotential reprogramming of fibroblasts in spheres.

In this study, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the epithelial-to-mesenchymal transition transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. Hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor.

Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice.

The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.

IKK1 control of epidermal differentiation is modulated by notch signaling.

The molecular mechanism by which IκB kinase 1 (IKK1) regulates epidermal differentiation and tumor suppression in the skin is not well understood. As two major regulatory signaling pathways that regulate epidermal homeostasis and differentiation, the p63 and Notch pathways were examined in Ikk1 mutant epidermis and keratinocytes. Ikk1 inactivation in keratinocytes resulted in increased p63 expression and repression of Notch signaling. The impaired differentiation of Ikk1(-/-) keratinocytes was partially rescued by overexpression of the active form of the Notch1 receptor, the Notch intracellular domain (NICD). In contrast, knockdown of p63 expression by RNA interference was unable to rescue the defect. These results suggest that, in the mammalian skin, IKK1 functions as a differentiation regulator and tumor suppressor through the Notch signaling pathway.

Notch signaling promotes the corneal epithelium wound healing.

PURPOSE: The Notch signaling pathway plays crucial roles in regulation of cell proliferation, differentiation and cell fate decision in multiple tissues and cell types. This study was designed to test the effects of enhanced Notch activity on corneal epithelium homeostasis and wound healing using the transgenic mice that overexpressed an activated Notch1 (NICD) in cornea epithelium. METHODS: The studies were performed on R26(fN1-ICD) transgenic mice that carry a NICD cDNA (cDNA) whose expression is prevented by a "Lox-STOP-Lox" cassette. When this transgenic mouse is bred to a mouse strain carrying a Cre recombinase expression cassette driven by a tissue-specific keratin 14 (K14) promoter, the floxed "STOP" cassette is excised and NICD is expressed in the cornea epithelium. The expression level of NICD and its downstream target genes, hairy and enhancer of split 1 (Hes1) and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), in the transgenic corneal epithelium was examined by quantitative PCR (qPCR). The phenotypes and morphology of the transgenic corneal epithelium were compared with that of wild type (WT) controls. The proliferation rate of the epithelial cells was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the differentiation statues were examined by K14, tumor protein p63 (p63), K12, and zona occludens 1 (ZO-1) immunoreactivity at either normal developmental condition or after corneal epithelial debridement. The corneal epithelial response to wound healing was studied by fluorescent staining and Richardson's staining macroscopically and by H&E staining at microscope level at 0, 6, 12, 18, and 24 h post injury. RESULTS: Although overexpression of NICD in cornea epithelium led to upregulation of its downstream targets, i.e., Hes1 and Hey1, this did not alter corneal epithelial cell proliferation and differentiation. However, wound healing induced Notch activity and overexpression of NICD promoted corneal epithelial wound healing, which was in agreement with more rapid early proliferation response in NICD transgenic mice than in the wild type control mice. CONCLUSIONS: These findings further demonstrate the functional role of Notch signaling in corneal epithelium wound healing response.

14-3-3σ and p63 play opposing roles in epidermal tumorigenesis.

14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using 'repeated epilation' heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.

Characterization and clinical evaluation of microsatellite instability and loss of heterozygosity within tumor-related genes in colorectal cancer.

BACKGROUND: Microsatellite instability (MSI) is a biomarker for better outcomes in colorectal cancer (CRC). However, this conclusion is controversial. In addition, MSs can be a useful marker for loss of heterozygosity (LOH) of genes, but this finding has not been well studied. Here, we aimed to clarify the predictive value of MSI/LOH within tumor-related genes in CRC. METHODS: We detected MSI/LOH of MSs in tumor-related genes and the Bethesda (B5) panel by STR scanning and cloning/sequencing. We further analyzed the relationship between MSI/LOH status and clinical features or outcomes by Pearson's Chi-square test, Fisher's exact test and the Kaplan-Meier method. RESULTS: The findings indicated that the MSI rates of B5 loci were all higher than those of loci in tumor-related genes. Interestingly, MSI/LOH of 2 loci in the B5 panel and 12 loci in tumor-related genes were associated with poorer outcomes, while MSI/LOH of the B5 panel failed to predict outcomes in CRC. MSI of BAT25, MSI/LOH of BAT26 and MSI of the B5 panel showed closer relationships with mucinous carcinoma. In addition, LOH-H of the B5 panel was associated with increased lymphatic metastasis. CONCLUSIONS: In summary, MSI/LOH of certain loci or the whole panel of B5 is related to clinical features, and several loci within tumor-related genes showed prognostic value in the outcomes of CRC.

14-3-3sigma controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway.

14-3-3sigma (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3sigma mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3sigma activity in corneal epithelial cells by overexpressing dominative negative 14-3-3sigma led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3sigma mutant-expressing corneal epithelial cells. We conclude that 14-3-3sigma is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts.

Purpose: Yap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs). Methods: Lens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism. Results: The heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/- LEC, and overexpression of Crim1 restored Yap1+/- LEC cell proliferation in vitro. Conclusions: Homozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.

NGF-induced Tyro3 and Axl function as survival factors for differentiating PC12 cells.

Tyro3 and Axl, two members of the TAM family of receptor tyrosine kinases, play important regulatory roles in a variety of tissues, including the central nervous, reproductive, immune, and vascular systems. We have found that expression of Tyro3 and Axl on PC12 cells is upregulated by nerve growth factor (NGF). PI3K inhibitor LY294002, which is known to inhibit NGF-induced PC12 differentiation, blocked up-regulation of Tyro3 and Axl. NGF regulates Tyro3 and Axl expression by activating their transcription. Both Tyro3 and Axl were associated with the NGF receptor, and protected PC12 cells from stress or toxin-induced cell death. Gas6, a common ligand for both Tyro3 and Axl, was able to replace NGF to support PC12 growth in serum-free medium, and to prevent cell death following serum deprivation. In summary, both Tyro3 and Axl receptors are upregulated by NGF on the differentiating PC12, where they collaborate with TrkA to support neuronal differentiation and survival.