Yi-Jen Hsueh

Chang Gung University, Taoyuan, Taiwan, Taiwan

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Publications (6)19.45 Total impact

  • Yi-Jen Hsueh, Pei-Ching Kuo, Jan-Kan Chen
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    ABSTRACT: The surface cells of corneal epithelium are regularly shed off and replaced by new cells that are derived from limbal epithelial stem cells (LESC). LESC are believed to reside in the basal layer of the limbal epithelium and are characterized with high expression levels of ΔNp63, a transcription factor which is believed to play roles in the regulation of LESC proliferation. In this study, we examined the transcriptional regulation of ΔNp63 in limbal epithelial cell. We employed DNA pull down assay followed by LC/MS analysis and cDNA microarray analysis to identify the transcription factors (TFs) that were capable of binding to ΔNp63 promoter or were expressed at higher levels in limbus over cornea. The TFs thus selected were further examined for their in vivo ΔNp63 promoter binding by ChIP assay. We identified six putative TFs (PAX6, EGR1, CEBPB, JUN, ATF3 and ARID5B) through the aforementioned approaches. Among them, PAX6 and EGR1 were shown to promote the transcription of ΔNp63 and led to increased cell proliferation. In contrast, CEBPB and ATF3 appeared to exert little or no effect on ΔNp63 expression, however, their silencing suppressed cell proliferation. Although JUN exhibited low promoter binding specificity, however, it affected ΔNp63 expression and limbal epithelial cell proliferation in ways similar to that of PAX6 and EGR1. Intriguingly, ARID5B was highly expressed in the limbal epithelial cell, however, its silencing by siRNA did not obviously affect the expression of ΔNp63, nor did it reduce cell proliferation of the limbal epithelial cell. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 07/2012; · 4.22 Impact Factor
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    ABSTRACT: To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin β1, and involucrin. Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin β1, and promotes the expression of involucrin. STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.
    Investigative ophthalmology & visual science 03/2011; 52(7):4685-93. · 3.43 Impact Factor
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    ABSTRACT: To examine the effects of TAp63 and DeltaNp63 on the proliferation and differentiation of rabbit limbal keratinocytes cultured on human amniotic membrane. Real-time Q-RT-PCR was used to quantify the relative abundance of TAp63 and DeltaNp63 transcripts in limbal, peripheral corneal, and central corneal epithelia. Antisense oligonucleotides were designed specifically to suppress the expression of TAp63 or DeltaNp63 in limbal keratinocytes, and their effects on cell proliferation and differentiation were examined. Immunofluorescence was used to examine the expressions of p63 and keratin-3 and -14. The expressions of TAp63 and DeltaNp63 transcripts appeared to be site specific. TAp63 was expressed at the highest level in limbus, decreased by approximately 10-fold in peripheral cornea and was undetectable in the central cornea. DeltaNp63 was also expressed at the highest level in limbus, decreased by approximately 35% in peripheral cornea, and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promoted differentiation. Suppression of DeltaNp63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. TAp63 and DeltaNp63 affect the proliferation of limbal keratinocytes by a different mechanism. The inhibition by TAp63 antisense oligos appeared to be secondary to the promotion of cell differentiation. In contrast, the inhibition by DeltaNp63 antisense oligos appeared to be independent of cell differentiation.
    Investigative Ophthalmology &amp Visual Science 10/2005; 46(9):3102-8. · 3.44 Impact Factor
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    ABSTRACT: Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IkappaB-alpha-NF-kappaB-TNF-alpha (tumor necrosis factor-alpha) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-alpha expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-alpha protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IkappaB-alpha phosphorylation and NF-kappaB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-kappaB activation and subsequently reduced TNF-alpha expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.
    Journal of Biomedical Science 11/2004; 11(6):773-80. · 2.46 Impact Factor
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    ABSTRACT: In this study, we examined the postnatal expression patterns of p63 and other keratinocyte stem cell markers in the rat cornea in an attempt to determine the markers that best represent characteristics of corneal keratinocyte stem cells. We show that the expression of p63 in the rat cornea is unique and differs from that observed in humans. It changes with age, from central cornea-positive, peripheral cornea-positive, and limbus-positive, to central cornea-positive, peripheral cornea-positive, and limbus-negative, and finally to central cornea-negative, peripheral cornea-positive, and limbus-negative, as examined by immunohistochemical staining. However, when a more sensitive staining method was used, the limbus was also shown to be positive for p63, indicating a lower level of expression than that of the peripheral cornea. The basal layer of the rat limbal epithelium is the site where beta-catenin+, K14+, PCNA-, and K3- cells reside. This cell layer is also the site where slow-cycling cells are located. In contrast with observations made in humans, our results clearly indicate that p63 is expressed in stem cells and young transient amplifying cells of the rat cornea, with higher levels of expression in the latter.
    Journal of Biomedical Science 07/2004; 11(5):641-51. · 2.46 Impact Factor
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    ABSTRACT: To describe the phenotypic characteristics of a limbal epithelial cell sheet outgrowth from a limbal explant cultured on amniotic membrane. Immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and the integrin alpha6/beta4 and alpha3/beta1 subunits in corneal and limbal tissues in a limbal explant and epithelial outgrowth cultured for 2 weeks on amniotic membrane. The expression patterns of p63, Ki-67, keratins, integrins, and connexin 43 in a limbal explant with an epithelial outgrowth cultured for 2 weeks on amniotic membrane resembled those in freshly prepared limbus. Moreover, the distribution of integrin subunits in positive cells of the limbal explant and its epithelial outgrowth was similar to that of the corneal epithelial cells during wound repair. The epithelial cell sheet grown from a limbal explant on amniotic membrane exhibited a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.
    Investigative Ophthalmology &amp Visual Science 11/2003; 44(11):4698-704. · 3.44 Impact Factor