Interaction of the Profilaggrin N-Terminal Domain with Loricrin in Human Cultured Keratinocytes and Epidermis

Abstract

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

Regulatory Role for the Profilaggrin N-Terminal Domain in Epidermal Homeostasis

“…In agreement with the results reported by Mildner et al, we did not observe profilaggrin knockdown altering the differentiation markers in the LSE cultures. In contrast, a recent study utilizing the raft culture keratinocyte system demonstrated significant loss of loricrin expression and some reduction in K10 expression as a consequence of an adenoviral vector-mediated profilaggrin silencing (Yoneda et al, 2012). Although adenoviral knockdown is expected to provide a longer-term knockdown of gene expression, similar to our lentiviral system, Yoneda et al did not observe epidermal thickening.…”

“…Nuclear localization of the profilaggrin N terminus has been observed to precede the event of keratinocytes losing their nuclei and transforming into corneocytes to form the stratum corneum (IshidaYamamoto et al, 1998), suggesting a critical role for this peptide in the later stages of terminal differentiation. Its involvement in differentiation is further supported by a recent study reporting the interactions with cornified envelopeassociated proteins and keratin 10 (Yoneda et al, 2012). Additional studies on the nuclear localization of profilaggrin N-terminal peptide were conducted using the transiently transfected keratinocytes in monolayer culture (Pearton et al, 2002).…”

Polycystic Kidney Disease with Steatocystoma Multiplex: Evidences for a Disruptive Effect of Mutated Polycystin-1 on Keratin 17 Polymerisation

“…At 48 h after nucleofection, cells were fixed with 4% paraformaldehyde and permeabilised using 0.1% Triton X-100 in phosphate-buffered saline. The cells were labelled and photographed as described previously (5,6). When HaCaT cells were doubly nucleofected with wild-type (WT) PKD1 and KRT17 genes, part of the PKD1 gene product looked filamentous, and this protein co-localised with K17 (Fig.…”

“…We also constructed an expression vector of KRT17 in the pcDNA3.1/V5-His vector (Life Technologies, Carlsbad, CA, USA). HaCaT cells were nucleofected using the Amaxa Cell Line Optimization Nucleofector Kit (Lonza, Walkersville, MD, USA), as previously described (5,6). At 48 h after nucleofection, cells were fixed with 4% paraformaldehyde and permeabilised using 0.1% Triton X-100 in phosphate-buffered saline.…”

Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27

“…We previously found PF-NT binds loricrin, a component of the cornified cell envelope (Yoneda et al , 2012). Similar to loricrin, stratifin (epithelial specific member of the 14-3-3 protein family also known as 14-3-3sigma) interacted with PF-NT and not with the CABD domain alone.…”