**8. The roles of other components of the aPKC-Par complex in hair loss**

#### **8.1. aPKC**

In the epidermis, aPKCζ, another isoform of aPKC, localizes in the cytoplasm and nucleus of basal cells, although its expression level is much lower than that of aPKCλ in the skin of newborns and adults (40-fold and 10-fold, respectively) [21]. Although the activity of aPKCζ in epithelial polarity in vitro is distinguishable from that of aPKCλ, mice lacking aPKCζ at the whole-body level were viable and showed no obvious skin phenotypes [60]. This may be attributed to the low expression of aPKCζ in the epidermis, and aPKCλ may compensate for the loss of aPKCζ. However, these results do not exclude the possibility that aPKCλ and aPKCζ synergistically regulate epithelial cell polarity, oriented cell division, epidermal differentiation, and HFSC maintenance. Indeed, the combined deletion of the aPKCλ/ι and aPKCζ isoforms in podocytes leads to defective glomerular maturation with incomplete capillary formation and mesangiolysis, and causes severe proteinuria and perinatal death [61]. Thus, studies on mutant mice with simultaneous epidermal inactivation of aPKCλ and aPKCζ would help provide further information on the synergism between the two.

#### **8.2. Junctional aPKC vs non-junctional aPKC**

Because aPKCs are localized to tight junctions, in aPKCλ cKO mice, the aPKCλ-Par6-Par3 complex at tight junctions was supposed to be absent or impaired in the granular layer. However, in the mutant mice, the overall multilayered architecture of the epidermis appeared to be normal, or rather hyperplasic [22, 23], suggesting that in contrast to simple epithelia, junctional aPKCλ is dispensable for establishing the polarity of the stratified epidermis, and that aPKCλ localized to the apical surface of basal cells during mitosis is more critical for maintaining epidermal homeostasis. Par6 and aPKCs form a stable heterodimer through their respective Phox/Bem1 (PB1) domains [7, 8], and aPKC-mediated phosphorylation is required for the dissociation of Par3 from the ternary complex. Thus, analysis of the dynamics of Par6 and Par3 or the Par6-Par3 complex in the absence of aPKCλ is helpful to understand the role of junctional aPKCλ.

The difference between junctional and non-junctional aPKC has been demonstrated by twostep chemical skin carcinogenesis experiments using epidermis-specific Par3 knockouts [62]. The epidermal loss of Par3 reduced papilloma formation and promoted keratoacanthoma formation, indicating that Par3 acts as a tumor promoter for papilloma and as a tumor suppressor for keratoacanthoma. In the absence of Par3, the aPKC-Par6 complex localized to the cytoplasm [62]. These results imply that the junctional aPKC-Par6 complex with Par3 is involved in papilloma formation, whereas the non-junctional, cytoplasmic aPKC-Par6 complex without Par3 is involved in keratoacanthoma formation.

#### **8.3. Par proteins**

The role of other components of the aPKC-Par complex (Par3 and Par6) in HFSC maintenance is unknown. Par3 is expressed throughout the interfollicular epidermis and the hair follicles, and it interacts with aPKCλ to colocalize at keratinocyte tight junctions [62]. However, no hair abnormalities were described in epidermis-specific Par3-deleted mice [62]. During SCD and ACD in the basal layer, Par3 was localized to the apical surface of the cell as a component of the Par3-LGN-inscuteable complex [51]. It would be useful to examine whether a shift from SCD to ACD in the hair follicle and subsequent HFSC depletion occurs in Par3-deficient mice as seen in aPKCλ cKO mice. Additionally, it would be interesting to investigate whether Par6 knockouts show similar phenotypes to those of aPKCλ knockouts. To the best of my knowledge, Par6 knockouts have not yet been reported. The presence of three isoforms of Par6 in mammals might make it difficult to reveal the phenotypes of Par6 inactivation.
