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Differential expression of papillomavirus L1 proteins encoded by authentic and codon modified L1 genes in methylcellulose-treated mouse keratinocytes.

Wang X, Li B, Zhao KN - Virol. J. (2007)

Bottom Line: Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs.Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs.These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Diamantina Institute for Cancer, Immunology & Metabolic Medicine, University of Queensland, Research Extension, Building 1, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia. xxxwang1@uq.edu.au

ABSTRACT
Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions. Here, we used methylcellulose to induce cell differentiation of primary mouse keratinocytes (KCs) in in vitro culture and assessed the expression of authentic and codon-modified version of L1 capsid genes from two PV types (HPV6b and BPV1). Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs. Western blot showed that methylcellulose significantly increased the levels of the L1 proteins expressed from two authentic L1 genes. Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs. These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.

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Cell morphology and expression of involucrin in the primary mouse KCs grown in KC-SF medium containing different concentration of methylcellulose for 2 days. (A). Gross cell morphology. Images ×200.(B). Immunofluorescence micrograph showing involucrin (green), β-tubulin (red) and nuclei (blue). Images ×400.
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Figure 1: Cell morphology and expression of involucrin in the primary mouse KCs grown in KC-SF medium containing different concentration of methylcellulose for 2 days. (A). Gross cell morphology. Images ×200.(B). Immunofluorescence micrograph showing involucrin (green), β-tubulin (red) and nuclei (blue). Images ×400.

Mentions: We prepared primary KCs from new-born mouse skin as previously described [8]. The primary mouse KCs were directly grown in semisolid KC-SF complete medium (Gibco, Australia) containing 0, 0.8% and 1.6% methylcellulose for two days. Resulting morphological changes of the cultured KCs after methylcellulose treatment were clearly observed, which included the changes of cell sizes and shapes (Fig 1A). The morphological changes of the cultured KCs were tightly associated with methylcellulose concentrations. The KCs changed their morphologies more dramatically in medium containing 1.6% methylcellulose than in medium containing 0.8% methylcellulose (Fig 1A). Immunofluorescence microscopy revealed further that the KCs grown in methylcellulose-free medium for two days showed weak involucrin signals, but expression of involucrin was significantly up-regulated in the KCs grown in medium containing methylcellulose (Fig. 1B). Methylcellulose treatment also resulted the KCs to change expression patterns of the other KC differentiation markers by reducing expression of basal keratins K14 and increasing expression of keratins K1 and K10 (data not shown). These data indicate that methylcellulose could induce mouse primary KCs to rapidly differentiate, consistent with previous observations for human KCs [13,14].


Differential expression of papillomavirus L1 proteins encoded by authentic and codon modified L1 genes in methylcellulose-treated mouse keratinocytes.

Wang X, Li B, Zhao KN - Virol. J. (2007)

Cell morphology and expression of involucrin in the primary mouse KCs grown in KC-SF medium containing different concentration of methylcellulose for 2 days. (A). Gross cell morphology. Images ×200.(B). Immunofluorescence micrograph showing involucrin (green), β-tubulin (red) and nuclei (blue). Images ×400.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2233611&req=5

Figure 1: Cell morphology and expression of involucrin in the primary mouse KCs grown in KC-SF medium containing different concentration of methylcellulose for 2 days. (A). Gross cell morphology. Images ×200.(B). Immunofluorescence micrograph showing involucrin (green), β-tubulin (red) and nuclei (blue). Images ×400.
Mentions: We prepared primary KCs from new-born mouse skin as previously described [8]. The primary mouse KCs were directly grown in semisolid KC-SF complete medium (Gibco, Australia) containing 0, 0.8% and 1.6% methylcellulose for two days. Resulting morphological changes of the cultured KCs after methylcellulose treatment were clearly observed, which included the changes of cell sizes and shapes (Fig 1A). The morphological changes of the cultured KCs were tightly associated with methylcellulose concentrations. The KCs changed their morphologies more dramatically in medium containing 1.6% methylcellulose than in medium containing 0.8% methylcellulose (Fig 1A). Immunofluorescence microscopy revealed further that the KCs grown in methylcellulose-free medium for two days showed weak involucrin signals, but expression of involucrin was significantly up-regulated in the KCs grown in medium containing methylcellulose (Fig. 1B). Methylcellulose treatment also resulted the KCs to change expression patterns of the other KC differentiation markers by reducing expression of basal keratins K14 and increasing expression of keratins K1 and K10 (data not shown). These data indicate that methylcellulose could induce mouse primary KCs to rapidly differentiate, consistent with previous observations for human KCs [13,14].

Bottom Line: Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs.Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs.These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Diamantina Institute for Cancer, Immunology & Metabolic Medicine, University of Queensland, Research Extension, Building 1, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia. xxxwang1@uq.edu.au

ABSTRACT
Papillomaviruses (PVs) are double-stranded DNA viruses that infect keratinocytes in differentiating epithelia and induce hyperproliferative lesions. Here, we used methylcellulose to induce cell differentiation of primary mouse keratinocytes (KCs) in in vitro culture and assessed the expression of authentic and codon-modified version of L1 capsid genes from two PV types (HPV6b and BPV1). Based on the quantitative RT-PCR analysis, methylcellulose treatment did not influence the transcriptional expression of both authentic and codon-modified L1 genes in KCs. Western blot showed that methylcellulose significantly increased the levels of the L1 proteins expressed from two authentic L1 genes. Conversely, methylcellulose dramatically decreased L1 protein expression in KCs transfected with two codon-modified L1 expression constructs. These data suggest that L1 protein expression is associated with KC differentiation induced by methylcellulose treatment and regulated at the post-transcriptional level.

Show MeSH
Related in: MedlinePlus