Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly. Wet AMD includes typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). The etiology and pathogenesis of CNV and PCV are not well understood. Genome-wide association studies have linked a multifunctional serine protease, HTRA1, to AMD. However, the precise role of HTRA1 in AMD remains elusive. By transgenically expressing human HTRA1 in mouse retinal pigment epithelium, we showed that increased HTRA1 induced cardinal features of PCV, including branching networks of choroidal vessels, polypoidal lesions, severe degeneration of the elastic laminae, and tunica media of choroidal vessels. In addition, HTRA1 mice displayed retinal pigment epithelium atrophy and photoreceptor degeneration. Senescent HTRA1 mice developed occult CNV, which likely resulted from the degradation of the elastic lamina of Bruch's membrane and up-regulation of VEGF. Our results indicate that increased HTRA1 is sufficient to cause PCV and is a significant risk factor for CNV.A dvanced age-related macular degeneration (AMD) can be classified into wet AMD and geographic atrophy (1, 2). Wet AMD includes the typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). CNV is caused by the growth of new blood vessels from the choroid into the subretinal pigment epithelium (RPE) and subretinal spaces, whereas PCV is caused by inner choroidal vessel abnormalities (3). PCV has two key features on indocyanine green angiography (ICGA): polypoidal vascular dilations and a network of branching abnormal choroid vessels (4). Both CNV and PCV can lead to recurrent serous exudation and hemorrhages (5). The etiology and pathogenesis of CNV and PCV are largely unknown.Numerous genetic association studies have shown that chromosome 10q26 is a major candidate region associated with the susceptibility of several types of AMD (6, 7), including PCV (8-10). The linkage peak was refined to two neighboring genes, HTRA1 (11, 12) and ARMS2 (or LOC387715) (13). HTRA1 is a multifunctional serine protease that is ubiquitously expressed in mammalian tissues (14, 15) but ARMS2 is primate-specific, with a proposed function in mitochondria (13, 16), extracellular matrix (17), or as a noncoding RNA (18). Variants in this region are in strong linkage disequilibrium (11-13, 16). There are three major competing hypotheses attributing increased risk of AMD to (i) increased HTRA1 (11, 12), (ii) decreased ARMS2 (16), or (iii) both increased HTRA1 and decreased ARMS2 (19). However, a series of studies on the influence of AMD-associated polymorphisms on the expression of ARMS2 and HTRA1 have yielded widely conflicting results (12,16,(18)(19)(20)(21)(22)(23)(24). As a result, the functional involvement of either HTRA1 or ARMS2 in AMD remains uncertain, despite strong genetic evidence (18,22). To clarify the role of HTRA1 in AMD pathogenesis, we transgenically expressed human HTRA1 in mouse RPE. We showed that increased HTRA1 is ...

SUMMARY The major types of non-small cell lung cancer (NSCLC) - squamous cell carcinoma and adenocarcinoma - have distinct immune microenvironments. We developed a genetic model of squamous NSCLC based on overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene expression and immune infiltrate features of human squamous NSCLC, including enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2–1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2–1, led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2–1 in SL mice (SNL) revealed that NKX2–1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage defining transcription factors determine the tumor immune microenvironment, which in turn may impact the nature of the tumor.

Changes in cancer cell identity can alter malignant potential and therapeutic response. Loss of the pulmonary lineage specifier NKX2-1 augments the growth of KRAS-driven lung adenocarcinoma and causes pulmonary to gastric transdifferentiation. Here, we show that the transcription factors FoxA1 and FoxA2 are required for initiation of mucinous NKX2-1-negative lung adenocarcinomas in the mouse and for activation of their gastric differentiation program. Foxa1/2 deletion severely impairs tumor initiation and causes a proximal shift in cellular identity, yielding tumors expressing markers of the squamocolumnar junction of the gastrointestinal tract. In contrast, we observe downregulation of FoxA1/2 expression in the squamous component of both murine and human lung adenosquamous carcinoma. Using sequential in vivo recombination, we find that FoxA1/2 loss in established KRAS-driven neoplasia originating from SPC-positive alveolar cells induces keratinizing squamous cell carcinomas. Thus, NKX2-1, FoxA1 and FoxA2 coordinately regulate the growth and identity of lung cancer in a context-specific manner.

It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin + Lrat −/− ) expressing, transgenically, short-wavelength-sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor's outer segment. In R-opsin's absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin's presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptidecompetition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling.rhodopsin | cone opsin | dimerization | protein trafficking R hodopsin and cone pigments mediate scotopic and photopic vision, respectively. They consist of opsin, the apo-protein, and 11-cis-retinal, a vitamin A-based chromophore. Light absorption by 11-cis-retinal triggers a conformational change in opsin, which in turn initiates a G protein-coupled receptor (GPCR) signaling pathway to lead to vision. Indeed, rhodopsin signaling is a prominent prototypical GPCR pathway from which a huge quantity of mechanistic details about such signaling in general has emerged. All along, it is a dogma that rhodopsin exists and functions as a monomer (1-6). About a decade ago, evidence began to emerge that rhodopsin may exist as a dimer, based on atomic force microscopy and cross-linking experiments performed on rod outersegment (ROS) disk membranes (7-9). However, this concept remains highly controversial because of the lack of in vivo evidence and also is puzzling because, unlike many GPCRs, monomeric rhodopsin is fully functional with respect to coupling to G protein (2, 4-6, 10) and to interactions with rhodopsin kinase and arrestin (11,12). In vivo evidence, albeit of paramount importance, is also challenging, because rhodopsin always exists as a single isoform in rod photoreceptors, thus making homomeric, higher-or...

Polypoidal choroidal vasculopathy (PCV) is a common subtype of wet age-related macular degeneration in Asian populations, whereas choroidal neovascularization is the typical subtype in Western populations. The cause of PCV is unknown. By comparing the phenotype of a PCV mouse model expressing protease high temperature requirement factor A1 (HTRA1) in retinal pigment epithelium with transgenic mice expressing the inactive HTRA1, we showed that HTRA1-mediated degradation of elastin in choroidal vessels is critical for the development of PCV, which exhibited destructive extracellular matrix remodeling and vascular smooth muscle cell loss. Compared with weak PCV, severe PCV exhibited prominent immune complex deposition, complement activation, and infiltration of inflammatory cells, suggesting inflammation plays a key role in PCV progression. More important, we validated these findings in human PCV specimens. Intravitreal delivery of an HTRA1 inhibitor (DPMFKLboroV) was effective (36% lesion reduction; P = 0.009) in preventing PCV initiation but ineffective in treating existing lesions. Anti-inflammatory glucocorticoid was effective in preventing PCV progression but ineffective in preventing PCV initiation. These results suggest that PCV pathogenesis occurs through two stages. The initiation stage is mediated by proteolytic degradation of extracellular matrix proteins attributable to increased HTRA1 activity, whereas the progression stage is driven by inflammatory cascades. This study provides a basis for understanding the differences between PCV and choroidal neovascularization, and helps guide the design of effective therapies for PCV.

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associated proteins including cone opsins (M- and S-opsins), cone transducin (Gαt2), G-protein-coupled receptor kinase 1 (GRK1) and guanylate cyclase 1 (GC1) has been suggested to play a role in cone degeneration. However, their precise role in cone degeneration is unclear. Here we investigated the role of S-opsin (Opn1sw) in cone degeneration in Lrat(-) (/-), a murine model for LCA, by genetic ablation of S-opsin. We show that deletion of just one allele of S-opsin from Lrat(-) (/-) mice is sufficient to prevent the rapid cone degeneration for at least 1 month. Deletion of both alleles of S-opsin prevents cone degeneration for an extended period (at least 12 months). This genetic prevention is accompanied by a reduction of endoplasmic reticulum (ER) stress in Lrat(-) (/-) photoreceptors. Despite cone survival in Opn1sw(-/-)Lrat(-) (/-) mice, cone membrane-associated proteins (e.g. Gαt2, GRK1 and GC1) continue to have trafficking problems. Our results suggest that cone opsins are the 'culprit' linking 11-cis-retinal deficiency to cone degeneration in LCA. This result has important implications for the current gene therapy strategy that emphasizes the need for a combinatorial therapy to both improve vision and slow photoreceptor degeneration.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.

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