Nancy Philp - Academia.edu (original) (raw)

Papers by Nancy Philp

Cytoskeleton, 1997

In several cell types, short-term increases in the concentration of the G-actin-sequestering pept... more In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of developing muscle cells.

American Journal of Physiology-cell Physiology, Dec 1, 2019

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the ret... more The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B ( Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b−/−, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b−/− mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

PubMed, Aug 1, 1987

We have examined the presence and distribution of integrin and fibronectin in the retinas of 21-d... more We have examined the presence and distribution of integrin and fibronectin in the retinas of 21-day chick embryos and adult rats, with particular emphasis on the question of localization in the retinal pigment epithelium (RPE). Isolated sheets of RPE solubilized and separated by gel electrophoresis contain integrin, as indicated by immunoblotting with polyclonal and monoclonal antibodies to the complex. By the same technique, antibodies to fibronectin reacted with a single protein in the isolated RPE. In both chick and rat, integrin and fibronectin were localized by indirect immunofluorescence exclusively to the basement membrane of the RPE, the choriocapillaris and the retinal-vitreal border. When isolated RPE cells from chick retinas were examined, integrin was seen to be present along the basolateral surfaces of the cells as well. Similarly, in the intact rat retina, staining for integrin could be seen along the lateral surfaces of some of the RPE cells. Neither integrin nor fibronectin were present along the apical surfaces of the RPE in either rat or chick. The close similarity between the location of integrin and fibronectin supports the idea that the RPE adheres to the basal lamina at least in part via integrin-fibronectin linkages. A clear implication of our results is that the adhesion between RPE and retina requires a different set of linkage proteins.

Progress in Retinal and Eye Research, Sep 1, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biochemical and Biophysical Research Communications, May 1, 1997

The retinal pigment epithelium transports lactate photoreceptor metabolism and viability (1,4,7).... more The retinal pigment epithelium transports lactate photoreceptor metabolism and viability (1,4,7). between two tissue compartments, the interphotore-Recently, we identified an integral membrane proceptor matrix and the choriocapillaris. In this report tein in the basolateral membrane of differentiated we describe a 2.45-kb cDNA isolated from a chick cDNA chick RPE cells (11). The protein was identified using RPE library that encodes a membrane protein found a monoclonal antibody (MAb 3C4) and called REMP only in RPE cells. The deduced protein has 542 amino (retinal epithelial membrane protein). REMP was acids with twelve putative membrane spanning dofound only in chick RPE cells and not in other chick mains. The cDNA has been designated MCT3 based on tissues such as neural retina, intestine, kidney or liver its 45% identity in amino acid sequence and structural (11). In this report, we describe the heterologous exsimilarity with the monocarboxylate transporters pression of a full length cDNA clone that encodes this MCT1 and MCT2. Stable transfectants (pCl-neo/MCT3), protein. The deduced amino-acid sequence of the cDNA made in a rat thyroid epithelial cell line (FRTL-5), ex-(isolated from a chick RPE expression library) is homolpress MCT3 RNA. Transfectants had enhanced pyrogous with the recently cloned monocarboxylate transuvate uptake (used as a measure of lactate uptake) porters MCT1 and MCT2 (12,13). The heterologous exwhich was proton-dependent and inhibited by a-cyano-4-hydroxycinnamate. In summary, MCT3's unique pression of a cDNA in FRTL-5 cells demonstrated that expression in RPE cells, multiple potential phosphory-REMP is a proton coupled monocarboxylate translation sites, and basolateral distribution suggest that porter and is designated MCT3. MCT3 may regulate lactate levels in the interphotoreceptor space.

Experimental Eye Research, Oct 1, 1998

MCT3 is a monocarboxylate transporter that is specifically expressed on the basolateral membrane ... more MCT3 is a monocarboxylate transporter that is specifically expressed on the basolateral membrane of retinal pigment epithelial cells (RPE). In these studies the temporal expression of MCT3 during ocular development was examined using Northern blot analysis. A 2.2 kb transcript (MCT3b) was detected in RPE by embryonic day 7 (E7) and was present throughout embryonic development. A 2.45 kb transcript (MCT3a) was expressed at low levels before E11 but its expression increased between E11 and E17. Using 5'-RACE (rapid amplification of cDNA ends) it has demonstrated that MCT3a and MCT3b mRNA had distinct 5'-untranslated sequences but shared the same translation start site. To determine the exon-intron structure and to understand the elements that control the tissue specific and developmental expression of MCT3, the MCT3 gene was cloned and sequenced from a chicken genomic library. The MCT3 gene is distributed over 8 kb of DNA and is composed of 6 exons. Coding sequences for MCT3 are found on exon 2 through exon 5. Comparison of the 5'-RACE sequence with the genomic sequence reveals that the two 5'-untranslated regions of the mRNAs are encoded by distinct exons, 1a and 1b, which are alternatively spliced to exon 2. These data suggest that two forms of MCT3 mRNAs could be generated by two distinct promoters that may be regulated in response to changes in the metabolic activity of the retina during development.

Proceedings of the National Academy of Sciences of the United States of America, Feb 11, 2019

American Journal of Physiology-regulatory Integrative and Comparative Physiology, Jun 1, 1998

Investigative Ophthalmology & Visual Science, Apr 1, 2003

PURPOSE. To evaluate the expression and subcellular distribution of proton-coupled monocarboxylat... more PURPOSE. To evaluate the expression and subcellular distribution of proton-coupled monocarboxylate transporters (MCTs) in human RPE in vivo and determine whether ARPE-19 cells retain the ability to express and differentially polarize these transporters. METHODS. Total RNA was prepared from human donor eyes and from ARPE-19 cell cultures. Expression of MCT transcripts was evaluated by RT-PCR amplification. Expression of MCT proteins in human RPE and ARPE-19 cells was evaluated by immunolocalization and Western blot analysis with isoform-specific anti-peptide antibodies. RESULTS. The expression of MCTs in human RPE was investigated by immunofluorescence analysis on frozen sections of human donor eyes. MCT1 antibody labeled the apical membrane of the RPE intensely, whereas MCT3 labeling was restricted to the basolateral membrane. MCT4 was detected in the neural retina but not in the RPE. ARPE-19 cells constitutively expressed MCT1 and MCT4 mRNAs. Expression of MCT3 mRNA increased over time as ARPE-19 cells established a differentiated phenotype. Western blot analysis revealed that ARPE-19 cells expressed high levels of MCT1 and MCT4 but very little MCT3 protein. Sections of differentiated ARPE-19 cells were labeled with MCT1, MCT4, and glucose transporter-1 antibodies. MCT1 was polarized to the apical membrane and MCT4 to the basolateral membrane, whereas GLUT1 was expressed in both membrane domains. CD147, which is necessary for targeting MCTs to the plasma membrane, was detected in the apical and basolateral membranes of human RPE in situ and ARPE-19 cells. CONCLUSIONS. These studies demonstrate for the first time that human RPE expresses two proton-coupled monocarboxylate transporters: MCT1 in the apical membrane and MCT3 in the basolateral membrane. The coordinated activities of these two transporters could facilitate the flux of lactate from the retina to the choroid. ARPE-19 cells express two MCT isoforms, polarized to different membrane domains: MCT1 to the apical membrane and MCT4 to the basolateral membrane. The polarized expression of MCTs in ARPE-19 demonstrates that these cells retain the cellular machinery necessary for transepithelial transport of lactate.

Genomics, Sep 1, 1999

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate... more Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.

Journal of Cell Biology, Aug 1, 1985

The retinal pigmented epithelium (RPE) is a simple cuboidal epithelium with apical processes whic... more The retinal pigmented epithelium (RPE) is a simple cuboidal epithelium with apical processes which, unlike many epithelia, do not extend freely into a lumen but rather interdigitate closely with the outer segments of the neural retina. To determine whether this close association was reflected in the cytoskeletal organization of the RPE, we studied the components of the cytoskeleton of the RPE and their localization in the body of the cell and in the apical processes. By relative mobility on SDS gels and by immunoblotting, we identified actin, vimentin, myosin, spectrin (240/235), and alpha-actinin as major components, and vinculin as a minor component. In addition, the RPE cytoskeleton contains polypeptides of Mr 280,000 and 250,000; the latter co-electrophoreses with actin-binding protein. By immunofluorescence, the terminal web region appeared similar to the comparable region of the intestinal epithelium that consists of broad belts of microfilaments containing myosin, actin, spectrin, and alpha-actinin. However, the components of the apical processes were very different from those of intestinal microvilli. We observed staining along the processes for myosin, actin, spectrin, alpha-actinin, and vinculin. The presence in the apical processes of contractile proteins and also of proteins typically found at sites of cell attachments suggests that the RPE may actively adhere to, and exert tension on, the neural retina.

Experimental Cell Research, Jul 1, 1995

Experimental Eye Research

Molecular & Cellular Proteomics, 2021

The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate pho... more The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1-embigin complex likely facilitates lactate transport through this cellular compartment. Highlights • The unique proteome of the photoreceptor outer segment plasma membrane is identified. • TMEM67, TMEM237, and embigin are novel unique components of this membrane. • Embigin in this membrane is associated with the monocarboxylate transporter MCT1. • The photoreceptor outer segment likely facilitates lactate transport.

Frontiers in Oncology, Jun 22, 2022

fibroblasts lacking MCT4 reduces tumor growth and decreases the expression of markers of metaboli... more fibroblasts lacking MCT4 reduces tumor growth and decreases the expression of markers of metabolic compartmentalization in tumors. In conclusion, metabolic compartmentalization with high expression of MCT4 in CAFs drives aggressiveness in ADT cancers.

Journal of Biological Chemistry, Jun 1, 2012

Background: microRNAs 204/211 regulate retinal pigment epithelial cell phenotype. Results: In RPE... more Background: microRNAs 204/211 regulate retinal pigment epithelial cell phenotype. Results: In RPE, MITF regulates miR-204/211 expression and down-regulation of MITF results in loss of RPE phenotype, which can be prevented by overexpressing miR-204/211. Conclusion: MITF-mediated expression of miR-204/211 directs RPE differentiation. Significance: miR-204/211-based therapeutics may be effective treatments for diseases that involve loss of RPE phenotype. The retinal pigment epithelium (RPE) plays a fundamental role in maintaining visual function and dedifferentiation of RPE contributes to the pathophysiology of several ocular diseases. To identify microRNAs (miRNAs) that may be involved in RPE differentiation, we compared the miRNA expression profiles of differentiated primary human fetal RPE (hfRPE) cells to dedifferentiated hfRPE cells. We found that miR-204/211, the two most highly expressed miRNAs in the RPE, were significantly down-regulated in dedifferentiated hfRPE cells. Importantly, transfection of pre-miR-204/211 into hfRPE cells promoted differentiation whereas adding miR-204/211 inhibitors led to their dedifferentiation. Microphthalmia-associated transcription factor (MITF) is a key regulator of RPE differentiation that was also down-regulated in dedifferentiated hfRPE cells. MITF knockdown decreased miR-204/211 expression and caused hfRPE dedifferentiation. Significantly, co-transfection of MITF siRNA with pre-miR-204/211 rescued RPE phenotype. Collectively, our data show that miR-204/211 promote RPE differentiation, suggesting that miR-204/211-based therapeutics may be effective treatments for diseases that involve RPE dedifferentiation such as proliferative vitreoretinopathy.

Figure S2 shows the effects of the antioxidant NAC on cell viability and MCT4 and TIGAR expressio... more Figure S2 shows the effects of the antioxidant NAC on cell viability and MCT4 and TIGAR expression in CTRL- and CSE-BJ1.

Figure S5 shows the tumor growth curves from both xenograft models, and the weights of tumors col... more Figure S5 shows the tumor growth curves from both xenograft models, and the weights of tumors collected at an early time point. It also shows immunohistochemistry images and quantification of CD45 and CD68 staining on FaDu tumors.