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Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis.
Mol Vis. 2003 Oct 15; 9:559-68.MV

Abstract

PURPOSE

The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis.

METHODS

PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K+ depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000TM reagent) with specific antisense oligonucleotides designed against the rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated.

RESULTS

Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs.

CONCLUSIONS

Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in rabbit conjunctival epithelial cells.

Authors+Show Affiliations

Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033-9121, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14566223

Citation

Qaddoumi, Mohamed G., et al. "Clathrin and Caveolin-1 Expression in Primary Pigmented Rabbit Conjunctival Epithelial Cells: Role in PLGA Nanoparticle Endocytosis." Molecular Vision, vol. 9, 2003, pp. 559-68.
Qaddoumi MG, Gukasyan HJ, Davda J, et al. Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis. Mol Vis. 2003;9:559-68.
Qaddoumi, M. G., Gukasyan, H. J., Davda, J., Labhasetwar, V., Kim, K. J., & Lee, V. H. (2003). Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis. Molecular Vision, 9, 559-68.
Qaddoumi MG, et al. Clathrin and Caveolin-1 Expression in Primary Pigmented Rabbit Conjunctival Epithelial Cells: Role in PLGA Nanoparticle Endocytosis. Mol Vis. 2003 Oct 15;9:559-68. PubMed PMID: 14566223.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis. AU - Qaddoumi,Mohamed G, AU - Gukasyan,Hovhannes J, AU - Davda,Jasmine, AU - Labhasetwar,Vinod, AU - Kim,Kwang-Jin, AU - Lee,Vincent H L, Y1 - 2003/10/15/ PY - 2003/10/21/pubmed PY - 2003/12/16/medline PY - 2003/10/21/entrez SP - 559 EP - 68 JF - Molecular vision JO - Mol. Vis. VL - 9 N2 - PURPOSE: The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis. METHODS: PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K+ depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000TM reagent) with specific antisense oligonucleotides designed against the rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated. RESULTS: Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs. CONCLUSIONS: Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in rabbit conjunctival epithelial cells. SN - 1090-0535 UR - https://www.unboundmedicine.com/medline/citation/14566223/Clathrin_and_caveolin_1_expression_in_primary_pigmented_rabbit_conjunctival_epithelial_cells:_role_in_PLGA_nanoparticle_endocytosis_ L2 - http://www.molvis.org/molvis/v9/a68/ DB - PRIME DP - Unbound Medicine ER -