Staphylococcus aureus Lipoteichoic Acid Inhibits Keratinocyte Differentiation Through a p63 Mediated Pathway

TO THE EDITOR

The bacterial pathogen, Staphylococcus aureus, frequently colonizes patients with Atopic Dermatitis (AD), a skin disease associated with loss of barrier function (Boguniewicz and Leung, 2011). Elevated levels of staphylococcal lipoteichoic acid (LTA) are found on the skin of patients affected by severe AD (Travers et al., 2010), however, the relative contributions of this product to keratinocyte differentiation and skin barrier formation are unknown.

To identify gene expression pathways altered by staphylococcal LTA, we performed gene microarray analysis on RNA isolated from primary human keratinocytes treated with media control or with LTA. We found keratinocytes are highly responsive to this bacterial product with 302 genes being either repressed or activated at least 2-fold (Supplementary Table S1). The biological process most significantly affected by LTA was that of epidermal development (Fig. 1a) with a 14.6-fold change. Other processes affected included the response to wounding, keratinocyte proliferation, negative regulation of cell differentiation and changes in the Notch signaling pathways. We focused our studies on examination of genes involved in the keratinocyte differentiation process. Fig. 1b and Supplementary Table S2 show that staphylococcal LTA down regulated a number of genes essential for keratinocyte differentiation, including KRT-1, KRT-10, and DSC-1 (Candi et al., 2005). The Notch ligands, JAG-2 and DLL-1, were similarly repressed. Interestingly, several genes involved in cell proliferation, including ID-1, FOS and Cyclin A1 were prominently up-regulated by LTA. To validate the results of the microarray, we measured expression of genes by real-time PCR, primarily focusing on genes known to play a critical role in the keratinocyte differentiation process and barrier formation. RT-PCR established that KRT-1, KRT-10, DSC-1, and DSG-1 mRNA levels were all significantly down-regulated by LTA in both undifferentiated and differentiated cells (Supplementary Fig. S1), while expression of the basal cell marker, KRT-5 remained unaffected. In summary, the results of our gene array profile demonstrate a role for staphylococcal LTA in inhibiting expression of genes critical for keratinocyte differentiation and barrier formation, while simultaneously inducing expression of genes involved in cell proliferation.

Fig. 1. Staphylococcus aureus LTA induces global changes in transcription that inhibit expression of genes involved in epidermal differentiation.

(a) Primary human keratinocytes were treated with media alone or 10 ug/ml LTA for 24 hours. Isolated RNA was subjected to microarray analysis. Multiple biological processes were significantly affected by treatment with staphylococcal LTA. (b) Microarray analysis shows that genes involved in keratinocyte differentiation were down regulated by LTA.

We were interested in determining the mechanism for LTA mediated regulation of gene transcription. Functioning upstream of other transcription factors, p63 has been described as the master regulator of skin development (Yang et al., 1999, Koster et al., 2007). p63 plays an essential role in maintaining self-renewing populations, and deficiency of p63 causes keratinocyte senescence (Keyes et al., 2005). In addition, p63 deficient keratinocytes have been reported to have defects in differentiation (Truong et al., 2006). However, the role of p63 in differentiation is controversial, as it has been shown that increasing the keratinocyte self-renewal capacity can restore differentiation in p63 knockout cells (Su et al., 2009). We used small interfering RNA (siRNA) to knock down p63 expression. Immunoblot analysis shows markedly reduced p63 protein levels (Supplementary Fig. S2). We found that keratinocytes transfected with p63 siRNA lost adherence and underwent significant cell death when trypsinized (Fig. 2a), consistent with previous studies (Carroll et al., 2006). However, we were able to maintain viability and attachment of p63 siRNA treated cells by leaving the keratinocytes untrypsinized (Fig. 2a). Adherent/untrypsinized keratinocytes transfected with p63 siRNA had reduced levels of the proliferation marker Ki67 (Supplementary Fig. S2) indicative of early cell senescence, as has previously been shown (Keyes et al., 2005). Due to premature cell death and adhesion defects, the study of differentiation in p63 deficient keratinocytes has been confounding. However, we found that adherent p63 deficient cells were able to differentiate similarly to controls (Fig. 2). We next examined the effects of LTA on control and p63 silenced cells. In control cells, LTA caused a significant reduction in expression of the differentiation and cohesion genes KRT-1, KRT-10, DSC-1, DSG-1 as well as significant increase in the cell proliferation marker, Cyclin A1 (Fig. 2b). In marked contrast, the inhibition of differentiation induced by LTA was blocked in p63 siRNA silenced cells (Fig. 2b), as was the increased expression of Cyclin A1. Therefore LTA mediated signals appear to proceed through p63 to activate or repress transcription of target genes. To confirm the results of gene expression, Western blots were performed to examine protein levels. We determined that LTA profoundly reduced levels of KRT-1, KRT-10, DSC-1, and DSG-1 protein in control cells. However, LTA mediated repression was reversed in p63 siRNA silenced cells (Fig. 2c). We conclude that LTA is a modulator of the activity of p63, converting p63 into an inhibitor of differentiation while simultaneously activating the proliferation response. To our knowledge this is the first report of a signaling pathway converging on p63 that inhibits the keratinocyte differentiation process.

Fig 2. LTA converts p63 into an inhibitor of keratinocyte differentiation.

(a) Keratinocytes transfected with control or p63 siRNA for 24 hours were either trypsinized and re-plated, or allowed to remain adherent. Photomicrographs show that p63 deficient keratinocytes remain adherent and viable in the absence trypsinization. Bar, 10µm (b) Keratinocytes were transfected with control or p63 siRNA for 24 hours. Cells were then induced to differentiate with calcium in the presence or absence of LTA. RT-PCR analysis of the indicated genes was performed on cells treated for one day (Cyclin A1), or four days (KRT-1, KRT-5, KRT-10, DSC-1, and DSG-1). Genes induced upon early keratinocyte differentiation were prominently down-regulated by LTA. Notably, the inhibition of differentiation induced by LTA is absent in p63 siRNA transfected cells. (c) Cells transfected with control or p63 siRNA were differentiated with calcium in the presence and absence of LTA. Following a four day incubation, cells were analyzed by Western blot for expression of the indicated proteins. Data are mean ± SEM, n = 3. **P<0.01; ***P<0.001 (LTA treated control vs. LTA treated p63 siRNA keratinocytes).

Notch signaling also directly regulates keratinocyte entry into differentiation (Nguyen et al., 2006, Rangarajan et al., 2001). Since Notch signaling is affected by LTA (Fig. 1), we next asked whether LTA mediated inhibition of gene expression was Notch dependent. We blocked Notch signaling with siRNA against ADAM10, as this protease is essential for Notch activation (Weber et al., 2011). Supplementary Fig. S3 shows that LTA mediated repression was similar in the presence and absence of ADAM10 siRNA. Therefore, inhibition of gene expression by LTA is not dependent on Notch.

In the current report, we show a unique role for staphylococcal LTA in repressing expression of a cluster of genes essential for keratinocyte cohesion, epidermal structure, and skin barrier function. The reduced expression of these proteins supports a role for LTA as a contributing factor in loss of barrier function in AD. We find that p63 contributes to this inhibition of differentiation, suggesting that p63 also may be a factor in AD. Increased levels of p63 have been shown in AD skin (Rizzo et al., 2016), as well as in epithelial tumors (King et al., 2008), correlating with increased proliferation and a block in differentiation signals. However, we demonstrate here that a second signal mediated by LTA is required for p63 to play an inhibitory role in keratinocyte differentiation. Our study suggests that p63 may be a novel target for modulation of keratinocyte differentiation in skin disease.

Abbreviations

AD

Atopic Dermatitis

ADAM

A Disintegrin and Metalloprotease

DLL

Delta-like

DSC

Desmocollin

DSG

Desmoglein

ID

Inhibitor of Differentiation/DNA binding

JAG

Jagged

Keratin

KRT

LTA

Lipoteichoic Acid

RT-PCR

Real Time PCR

siRNA

small interfering RNA