Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer (PCa) cells and demonstrated that it promotes PCa progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1–positive (Cav-1+) HGPIN and Cav-1+ primary PCa (rho = 0.655, P< 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and PCa in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR2PB promoter. In this ARR2PB–c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and PCa cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1–negative epithelia adjacent to those lesions (10.02 vs 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1–induced PCa cell survival and positive-feedback regulation of cellular Cav-1 levels, respectively. In multiple PCa cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc–induced Cav-1 overexpression, suggesting a c-Myc–VEGF–Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and PCa. Further, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1+ primary PCa.
Caveolin-1; c-Myc; prostatic intraepithelial neoplasia; prostate cancer; metaplasia
Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and Gαi, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the Gαi binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.
ELL-associated factor 2 (EAF2) is an androgen-responsive tumor suppressor frequently deleted in advanced prostate cancer that functions as a transcription elongation factor of RNA Pol II through interaction with the ELL family proteins. EAF2 knockout mice on a 129P2/OLA-C57BL/6J background developed late-onset lung adenocarcinoma, hepatocellular carcinoma, B-cell lymphoma and high-grade prostatic intraepithelial neoplasia. In order to further characterize the role of EAF2 in the development of prostatic defects, the effects of EAF2 loss were compared in different murine strains. In the current study, aged EAF2−/− mice on both the C57BL/6J and FVB/NJ backgrounds exhibited mPIN lesions as previously reported on a 129P2/OLA-C57BL/6J background. In contrast to the 129P2/OLA-C57BL/6J mixed genetic background, the mPIN lesions in C57BL/6J and FVB/NJ EAF2−/− mice were associated with stromal defects characteristic of a reactive stroma and a statistically significant increase in prostate microvessel density. Stromal inflammation and increased microvessel density was evident in EAF2-deficient mice on a pure C57BL/6J background at an early age and preceded the development of the histologic epithelial hyperplasia and neoplasia found in the prostates of older EAF2−/− animals. Mice deficient in EAF2 had an increased recovery rate and a decreased overall response to the effects of androgen deprivation. EAF2 expression in human cancer was significantly down-regulated and microvessel density was significantly increased compared to matched normal prostate tissue; furthermore EAF2 expression was negatively correlated with microvessel density. These results suggest that the EAF2 knockout mouse on the C57BL/6J and FVB/NJ genetic backgrounds provides a model of PIN lesions associated with an altered prostate microvasculature and reactive stromal compartment corresponding to that reported in human prostate tumors.
The incidence and mortality of prostate cancer (PCa) vary greatly in different geographic regions, for which lifestyle factors, such as dietary fat intake, have been implicated. Human 15-lipoxygenase-1 (h15-LO-1), which metabolizes polyunsaturated fatty acids, is a highly regulated, tissue-specific, lipid-peroxidating enzyme that functions in physiological membrane remodeling and in the pathogenesis of atherosclerosis, inflammation, and carcinogenesis. We have shown that aberrant overexpression of 15-LO-1 occurs in human PCa, particularly high-grade PCa, and in high-grade prostatic intraepithelial neoplasia (HGPIN), and that the murine orthologue is increased in SV40-based genetically engineered mouse (GEM) models of PCa, such as LADY and TRansgenic Adenocarcinoma of Mouse Prostate. To further define the role of 15-LO-1 in prostate carcinogenesis, we established a novel GEM model with targeted overexpression of h15-LO-1 in the prostate [human fifteen lipoxygenase-1 in mouse prostate (FLiMP)]. We used a Cre- mediated and a loxP-mediated recombination strategy to target h15-LO-1 specifically to the prostate of C57BL/6 mice. Wild-type (wt), FLiMP+/-, and FLiMP+/+ mice aged 7 to 21, 24 to 28, and 35 weeks were characterized by histopathology, immunohistochemistry (IHC), and DNA/RNA and enzyme analyses. Compared to wt mice, h15-LO-1 enzyme activity was increased similarly in both homozygous FLiMP+/+ and hemizygous FLiMP+/- prostates. Dorsolateral and ventral prostates of FLiMP mice showed focal and progressive epithelial hyperplasia with nuclear atypia, indicative of the definition of mouse prostatic intraepithelial neoplasia (mPIN) according to the National Cancer Institute. These foci showed increased proliferation by Ki-67 IHC. No progression to invasive PCa was noted up to 35 weeks. By IHC, h15-LO-1 expression was limited to luminal epithelial cells, with increased expression in mPIN foci (similar to human HGPIN). In summary, targeted overexpression of h15-LO-1 (a gene overexpressed in human PCa and HGPIN) to mouse prostate is sufficient to promote epithelial proliferation and mPIN development. These results support 15-LO-1 as having a role in prostate tumor initiation and as an early target for dietary or other prevention strategies. The FLiMP mouse model should also be useful in crosses with other GEM models to further define the combinations of molecular alterations necessary for PCa progression.
Transgenic; mouse model; genetically engineered mouse model; arachidonic acid; prostate cancer
Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. Id4 is expressed in the normal prostate where its expression is also regulated by androgens. In this study we investigated the effect of loss of Id4 (Id4-/-) on adult prostate morphology.
Histological analysis was performed on prostates from 6-8 weeks old Id4-/-, Id4+/- and Id4+/+ mice. Expression of Id1, Sox9, Myc, androgen receptor, Akt, p-Akt, Pten and Nkx3.1 was investigated by immunohistochemistry. Androgen receptor binding on NKX3.1 promoter was studied by chromatin immuno-precipitation. Id4 was either over-expressed or silenced in prostate cancer cell lines DU145 and LNCaP respectively followed by analysis of PTEN, NKX3.1 and Sox9 expression.
Id4-/- mice had smaller prostates with fewer tubules, smaller tubule diameters and subtle mPIN like lesions. Levels of androgen receptor were similar between wild type and Id4-/- prostate. Decreased NKX3.1 expression was in part due to decreased androgen receptor binding on NKX3.1 promoter in Id4-/- mice. The increase in the expression of Myc, Sox9, Id1, Ki67 and decrease in the expression of PTEN, Akt and phospho-AKT was associated with subtle mPIN like lesions in Id4-/- prostates. Finally, prostate cancer cell line models in which Id4 was either silenced or over-expressed confirmed that Id4 regulates NKX3.1, Sox9 and PTEN.
Our results suggest that loss of Id4 attenuates normal prostate development and promotes hyperplasia/dysplasia with subtle mPIN like lesions characterized by gain of Myc and Id1 and loss of Nkx3.1 and Pten expression. One of the mechanisms by which Id4 may regulate normal prostate development is through regulating androgen receptor binding to respective response elements such as those on NKX3.1 promoter. In spite of these complex alterations, large neoplastic lesions in Id4-/- prostates were not observed suggesting the possibility of mechanisms/pathways such as loss of Akt that could restrain the formation of significant pre-cancerous lesions.
Id4; Prostate; Androgen receptor; NKX3.1; Akt; PTEN
The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Gαi; in addition, Gβ13F is also required. However, the role of Gαi and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Gαi mutants and show that Gαi and Gβ13F play distinct roles. Gαi is required for Pins to localize to the cortex, and the effects of loss of Gαi or pins are highly similar, supporting the idea that Pins/Gαi act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gβ13F appears to regulate the asymmetric localization/stability of all apical components, and Gβ13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.
neuroblast; asymmetric division; astral microtubules; heterotrimeric G proteins; Drosophila
The scaffolding protein Canoe regulates spindle orientation by binding to RanGTP and recruiting RanGTP and Mud to the cell cortex.
Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila melanogaster neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. In this paper, we use our recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. We show that a previously uncharacterized portion of Canoe directly binds the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe–PinsTPR interaction recruits Canoe to the cell cortex and is required for activation of the PinsTPR-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. We show that the Canoe Ras-association (RA) domains directly bind RanGTP and that both the CanoeRA domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway.
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here we develop a method to construct cortical polarity in a normally unpolarized cell line, and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals)-dependent spindle orientation. We identify a previously unrecognized evolutionarily-conserved Pins domain (PinsLINKER) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized PinsTPR domain has no function alone, but placing the PinsTPR in cis to the PinsLINKER gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.
Auxin is a key regulator of plant development and its differential distribution in plant tissues, established by a polar cell to cell transport, can trigger a wide range of developmental processes. A few members of the two families of auxin efflux transport proteins, PIN-formed (PIN) and P-glycoprotein (ABCB/PGP), have so far been characterized in maize. Nine new Zea mays auxin efflux carriers PIN family members and two maize PIN-like genes have now been identified. Four members of PIN1 (named ZmPIN1a–d) cluster, one gene homologous to AtPIN2 (ZmPIN2), three orthologs of PIN5 (ZmPIN5a–c), one gene paired with AtPIN8 (ZmPIN8), and three monocot-specific PINs (ZmPIN9, ZmPIN10a, and ZmPIN10b) were cloned and the phylogenetic relationships between early-land plants, monocots, and eudicots PIN proteins investigated, including the new maize PIN proteins. Tissue-specific expression patterns of the 12 maize PIN genes, 2 PIN-like genes and ZmABCB1, an ABCB auxin efflux carrier, were analyzed together with protein localization and auxin accumulation patterns in normal conditions and in response to drug applications. ZmPIN gene transcripts have overlapping expression domains in the root apex, during male and female inflorescence differentiation and kernel development. However, some PIN family members have specific tissue localization: ZmPIN1d transcript marks the L1 layer of the shoot apical meristem and inflorescence meristem during the flowering transition and the monocot-specific ZmPIN9 is expressed in the root endodermis and pericycle. The phylogenetic and gene structure analyses together with the expression pattern of the ZmPIN gene family indicate that subfunctionalization of some maize PINs can be associated to the differentiation and development of monocot-specific organs and tissues and might have occurred after the divergence between dicots and monocots.
PIN-formed; auxin efflux carriers; kernel development; inflorescences; monocots; polar auxin transport; Zea mays
Human tumors are heterogeneous and evolve through a dynamic process of genetic mutation and selection. During this process, the effects of a specific mutation on the incipient cancer cell may dictate the nature of subsequent mutations that can be tolerated or selected for, affecting the rate at which subsequent mutations occur. Here we have used a new mouse model of prostate cancer that recapitulates several salient features of the human disease to examine the relative rates in which the remaining wild type alleles of Pten and p53 tumor suppressor genes are lost. In this model, focal overexpression of c-MYC in a few prostate luminal epithelial cells provokes a mild proliferative response. In the context of compound Pten/p53 heterozygosity, c-MYC-initiated cells progress to prostatic intraepithelial neoplasia (mPIN) and adenocarcinoma lesions with marked heterogeneity within the same prostate glands. Using Laser Capture Microdissection and gene copy number analyses, we found that the frequency of Pten loss was significantly higher than that of p53 loss in mPIN but not invasive carcinoma lesions. c-MYC overexpression, unlike Pten loss, did not activate the p53 pathway in transgenic mouse prostate cells, explaining the lack of selective pressure to lose p53 in the c-MYC-overexpressing cells. This model of heterogeneous prostate cancer based on alterations in genes relevant to the human disease may be useful for understanding pathogenesis of the disease and testing new therapeutic agents.
c-MYC; Pten; p53; prostate cancer; rate of mutations
The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.
Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.
Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical–basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ13F to the membrane, indicating an essential role of cortical Gγ1–Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins–Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka–atypical PKC–Par6–Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable–Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.
epithelium; cell polarity; heterotrimeric G protein; spindle orientation; Drosophila melanogaster
Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms2–7. Polar PIN localization determines the direction of intercellular auxin flow8, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.
Previous studies have shown that the Ron receptor is overexpressed in prostate cancer and Ron expression increases with disease severity in humans and the mouse TRAMP model. Here, the causal role of Ron overexpression in the murine prostate was examined in the development and progression of prostate cancer. Transgenic mouse strains were generated which selectively overexpressed Ron in the prostate epithelium and prostate histopathology was evaluated and compared to wild type controls. Ron overexpression led to the development of prostate intraepithelial neoplasia (mPIN) with local invasion and was associated with increases in prostate cell proliferation and decreases in cell death.
Ron receptor; prostate cancer; mouse prostate intraepithelial neoplasia; receptor tyrosine kinase; Met receptor
Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.
Methodology and Principle Findings
Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.
Conclusions and Significance
Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.
The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn–enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.
PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.
Auxin is a unique plant hormone, which is actively and directionally transported in plant tissues. Transported auxin locally accumulates in the plant body and triggers a multitude of responses, including organ formation and patterning. Therefore, regulation of the directional auxin transport is very important in multiple aspects of plant development. The PIN-FORMED (PIN) family of auxin transporters is known to localize at specific sides of cells and export auxin from the cells, enabling the directional transport of auxin in the tissues. PIN proteins are rapidly shuttling between the plasma membrane and intracellular compartments, potentially allowing dynamic changes of the asymmetric localization according to developmental and environmental cues. Here, we discovered that a mutation in the Sec1/Munc18 family protein VPS45 abolishes its own early endosomal localization and compromises intracellular trafficking of PIN proteins. By genetic and pharmacological inhibition of early endosomal trafficking, we also revealed that another early endosomal protein, ARF GEF BEN1, is involved in early endosomal trafficking at a distinct step. Furthermore, we showed that these components play crucial roles in polar localization and dynamic repolarization of PIN proteins, which underpin various developmental processes. These findings highlight the indispensable roles of early endosomal components in regulating PIN polarity and plant architecture.
Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6.
The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR.
In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens.
Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation.
Phosphorylation on Ser/Thr-Pro motifs is a major mechanism regulating many events involved in cell proliferation and transformation, including centrosome duplication, whose defects have been implicated in oncogenesis. Certain phosphorylated Ser/Thr-Pro motifs can exist in two distinct conformations whose conversion in certain proteins is catalyzed specifically by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and is important for the activation of multiple oncogenic pathways, and its deletion suppresses the ability of certain oncogenes to induce cancer in mice. However, little is known about the role of Pin1 in centrosome duplication and the significance of Pin1 overexpression in cancer development in vivo. Here we show that Pin1 overexpression correlates with centrosome amplification in human breast cancer tissues. Furthermore, Pin1 localizes to and copurifies with centrosomes in interphase but not mitotic cells. Moreover, Pin1 ablation in mouse embryonic fibroblasts drastically delays centrosome duplication without affecting DNA synthesis and Pin1 inhibition also suppresses centrosome amplification in S-arrested CHO cells. In contrast, overexpression of Pin1 drives centrosome duplication and accumulation, resulting in chromosome missegregation, aneuploidy, and transformation in nontransformed NIH 3T3 cells. More importantly, transgenic overexpression of Pin1 in mouse mammary glands also potently induces centrosome amplification, eventually leading to mammary hyperplasia and malignant mammary tumors with overamplified centrosomes. These results demonstrate for the first time that the phosphorylation-specific isomerase Pin1 regulates centrosome duplication and its deregulation can induce centrosome amplification, chromosome instability, and oncogenesis.
The Wnt/β-catenin pathway promotes proliferation of neural progenitor cells (NPCs) at early stages and induces neuronal differentiation from NPCs at late stages, but the molecular mechanisms that control this stage-specific response are unclear. Pin1 is a prolyl isomerase that regulates cell signaling uniquely by controlling protein conformation after phosphorylation, but its role in neuronal differentiation is not known. Here we found that whereas Pin1 depletion suppresses neuronal differentiation, Pin1 overexpression enhances it, without any effects on gliogenesis from NPCs in vitro. Consequently, Pin1-null mice have significantly fewer upper layer neurons in the motor cortex and severely impaired motor activity during the neonatal stage. A proteomic approach identified β-catenin as a major substrate for Pin1 in NPCs, in which Pin1 stabilizes β-catenin. As a result, Pin1 knockout leads to reduced β-catenin during differentiation but not proliferation of NPCs in developing brains. Importantly, defective neuronal differentiation in Pin1 knockout NPCs is fully rescued in vitro by overexpression of β-catenin but not a β-catenin mutant that fails to act as a Pin1 substrate. These results show that Pin1 is a novel regulator of NPC differentiation by acting on β-catenin and provides a new postphosphorylation signaling mechanism to regulate developmental stage-specific functioning of β-catenin signaling in neuronal differentiation.
A study in leaf epidermal pavement cells reveals that auxin activation of a Rho-like GTPase from plants induces inhibition of endocytosis through the clathrin-mediated pathway by regulating the accumulation of cortical F-actin.
Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.
Formation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long-distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of “puzzle-piece” polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also important for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocytosis is a common mechanism for cell polarization in multicellular organisms.
The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC.
MAEC, transduced with lentivirus encoding an inactive control short hairpin (sh) RNA or one targeting PIN1 that reduced PIN1 by 85%, were used. Cells were treated with LPS/IFN, calpain inhibitors (carbobenzoxy-valinyl-phenylalaninal (zVF), PD150606), cycloheximide and COX inhibitors to determine the effect of PIN1 depletion on COX-2 and calpain.
LPS or IFN alone did not induce COX-2. However, treatment with 10 μg LPS plus 20 ng IFN per ml induced COX-2 protein 10-fold in Control shRNA MAEC. Induction was significantly greater (47-fold) in PIN1 shRNA cells. COX-2-dependent prostaglandin E2 production increased 3-fold in KD MAEC, but did not increase in Control cells. The additional increase in COX-2 protein due to PIN1 depletion was post-transcriptional, as induction of COX-2 mRNA by LPS/IFN was the same in cells containing or lacking PIN1. Instead, the loss of COX-2 protein, after treatment with cycloheximide to block protein synthesis, was reduced in cells lacking PIN1 in comparison with Control cells, indicating that degradation of the enzyme was reduced. zVF and PD150606 each enhanced the induction of COX-2 by LPS/IFN. zVF also slowed the loss of COX-2 after treatment with cycloheximide, and COX-2 was degraded by exogenous μ-calpain in vitro. In contrast to iNOS, physical interaction between COX-2 and PIN1 was not detected, suggesting that effects of PIN1 on calpain, rather than COX-2 itself, affect COX-2 degradation. While cathepsin activity was unaltered, depletion of PIN1 reduced calpain activity by 55% in comparison with Control shRNA cells.
PIN1 reduced calpain activity and slowed the degradation of COX-2 in MAEC, an effect recapitulated by an inhibitor of calpain. Given the sensitivity of COX-2 and iNOS to calpain, PIN1 may normally limit induction of these and other calpain substrates by maintaining calpain activity in endothelial cells.
Pin1 isomerizes the phosphorylated Ser/Thr-Pro peptide bonds and regulates the functions of its binding proteins by inducing conformational changes. Involvement of Pin1 in the aging process has been suggested based on the phenotype of Pin1-knockout mice and its interaction with lifespan regulator protein, p66Shc. In this study, we utilize a proteomic approach and identify peroxiredoxin 1 (PRDX1), another regulator of aging, as a novel Pin1 binding protein. Pin1 binds to PRDX1 through interacting with the phospho-Thr90-Pro91 motif of PRDX1, and this interaction is abolished when the Thr90 of PRDX1 is mutated. The Pin1 binding motif, Thr-Pro, is conserved in the 2-Cys PRDXs, PRDX1–4 and the interactions between Pin1 and PRDX2–4 are also demonstrated. An increase in hydrogen peroxide buildup and a decrease in the peroxidase activity of 2-Cys PRDXs were observed in Pin1−/− mouse embryonic fibroblasts (MEFs), with the activity of PRDXs restored when Pin1 was re-introduced into the cells. Phosphorylation of PRDX1 at Thr90 has been shown to inhibit its peroxidase activity; however, how exactly the activity of PRDX1 is regulated by phosphorylation still remains unknown. Here, we demonstrate that Pin1 facilitates the protein phosphatase 2A-mediated dephosphorylation of PRDX1, which helps to explain the accumulation of the inactive phosphorylated form of PRDX1 in Pin1−/− MEFs. Collectively, we identify Pin1 as a novel PRDX1 binding protein and propose a mechanism for Pin1 in regulating the metabolism of reactive oxygen species in cells.
Pin1; PRDX1; p66Shc; PP2A; phosphorylation; aging and reactive oxygen species