Peptidoglycan recognition protein 2 (N-acetylmuramoyl-L-Ala amidase) is induced in keratinocytes by bacteria through the p38 kinase pathway

Abstract

Human peptidoglycan recognition protein 2 (PGLYRP2) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and is constitutively produced in the liver and secreted into the blood. Here we demonstrate that PGLYRP2 was not expressed in healthy human skin and had low expression in the eye. However, upon exposure to gram-positive and gram-negative bacteria or cytokines, PGLYRP2 expression was highly induced in keratinocytes and to a lower level in corneal epithelial cells. Expression of PGLYRP2 was not induced in nonepithelial cells. Exposure of keratinocytes to bacteria induced keratinocyte differentiation and stress response and inhibited activation of signal transduction molecules involved in cell proliferation. Induction of PGLYRP2 expression correlated with expression of differentiation markers (cytokeratins and transglutaminase). Bacteria induced activation of p38 mitogen-activated protein kinase (MAPK) in keratinocytes, which was required for the induction of PGLYRP2 expression, because induction of PGLYRP2 transcription by bacteria was inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by a dominant-negative p38 construct. Induction of PGLYRP2 expression by bacteria (in contrast to expression of human beta-defensin-2) was not mediated by Toll-like receptor 2 or 4. PGLYRP2 may function in the skin and the eyes as an inducible scavenger of proinflammatory peptidoglycan.

FIG. 1.

Bacteria induce expression of PGLYRP2 mRNA in human keratinocytes and corneal epithelial cells, shown by real-time RT-PCR (A to E) and Northern blotting (F). (A) Constitutive PGLYRP2 mRNA expression in normal human liver, skin, cornea, and sclera. (B) Induction of PGLYRP2 mRNA by bacteria in cultured human epidermal keratinocytes and human corneal epithelial cells (HCEC) but not in HUVEC, U373 astrocyte cell line, and peripheral blood monocytes. (C) Dose response and (D) time course of induction of PGLYRP2 mRNA expression by B. subtilis and E. cloacae in keratinocytes. Nil, no stimulus. (E) PGLYRP2 mRNA expression in keratinocytes is induced by a variety of bacteria, a yeast, and IL-1β and TNF-α. (F) Induction of 2-kb PGLYRP2 mRNA by B. subtilis and E. cloacae in keratinocytes. The results are means of three to four experiments ± standard errors (A, B, D, and E) or the averages of two experiments (C) or one out of two similar experiments (F).

FIG. 2.

Bacteria induce expression of PGLYRP2 protein in keratinocytes, which correlates with high expression of cytokeratins and transglutaminase. Keratinocytes were cultured in medium alone or with B. subtilis as indicated, and the expression of PGLYRP2 and cytokeratins or transglutaminase was determined by two-color IF. The results represent one out of two to three similar experiments. Similar results were obtained for keratinocyte cultures stimulated with E. cloacae (not shown).

FIG. 3.

Stimulation of keratinocytes with bacteria induces phosphorylation of p38 MAPK. Keratinocytes were cultured in medium alone or were stimulated with B. subtilis or E. cloacae, and the presence of phosphorylated p38 MAPK (P-p38) or total p38 MAPK (p38) in the cell lysates was determined on Western blots. The results are a representative blot (A) and quantification of average increases of P-p38 (expressed as the ratio to unstimulated time 0 group; means from four experiments).

FIG. 4.

Activation of p38 MAPK by bacteria is required for induction PGLYRP2 expression. (A) Induction PGLYRP2 expression in keratinocytes by B. subtilis or E. cloacae is inhibited by an inhibitor of p38 MAPK activation (SB203580) but not by inhibitors of ERK1 and ERK2 activation (PD98059), JNK activation (SP600125), and PI3 kinase activation (wortmannin). The results represent the means of four experiments ± standard errors (*, P ≤ 0.005 [treated versus untreated or dimethyl sulfoxide control]); all other differences were not significant (P > 0.05). DMSO, dimethyl sulfoxide. (B) Induction of transcription of PGLYRP2 promoter by bacteria in transiently transfected keratinocytes is inhibited by dominant-negative p38 (DNp38) but not by dominant-negative ERK (DNERK). The results represent means for four cultures from two experiments (*, P ≤ 0.015) (DNp38 versus control vector [CV]).

FIG. 5.

PGLYRP2, in contrast to HBD2, is not induced through TLR2 or TLR4. (A and B) Induction of PGLYRP2 mRNA expression in keratinocytes by B. subtilis or E. cloacae (A) is not inhibited by anti-TLR2 and anti-TLR4 antibodies, in contrast to HBD2 mRNA expression (B), which is significantly inhibited by anti-TLR2 antibodies or by a mixture of anti-TLR2 and anti-TLR4 antibodies (following stimulation with B. subtilis or E. cloacae, respectively). The results in panels A and B are means of four experiments; P values versus the IgG control are shown, and all other differences were not significant (P > 0.05). (C) Induction of transcription of PGLYRP2 promoter in transiently transfected keratinocytes following stimulation with B. subtilis or E. cloacae is not increased by cotransfection with TLR2 or TLR4. (D) Transcription of PGLYRP2 promoter in transiently transfected 293 cells following stimulation with B. subtilis or E. cloacae is not induced by cotransfection with TLR2 or TLR4, in contrast to transcription of the HBD2 promoter, which is induced by both B. subtilis and E. cloacae or by E. cloacae only (but not B. subtilis) in 293 cells transfected with TLR2 or TLR4, respectively. TLR2-dependent induction of HBD2 transcription by B. subtilis or E. cloacae is inhibited by dominant-negative (DN) IκB (DN-IκB), DN-MyD88, and DN-IRAK1, but not by DN-IRAK2. All cells transfected with TLR2 or TLR4 were also cotransfected with CD14 or CD14 plus MD-2, respectively. The results in panels C and D are means of three experiments. Nil, no stimulus.