**2. Progressive hair loss**

receptors, and tyrosine kinase receptors through lipid-derived secondary messengers [1]. The PKC family members share a highly conserved carboxy-terminal kinase domain, and differences in their requirements for lipids and calcium for activation are attributed to structural differences in the amino-terminal regulatory domain [2]. In mammals, the PKC family is composed of the following three structurally and functionally distinct subgroups: conventional PKCs (cPKC; α, βI/II, and γ), novel PKCs (nPKC; δ, ε, η, and θ), and atypical PKCs (aPKC; ζ and ι/λ: λ in mice) (**Figure 1**) [2, 3]. The cPKCs have a prototypic regulatory domain consisting of the following two conserved regions: C1 and C2. The C1 region serves as a binding site for diacylglycerol (DAG) and phospholipids, whereas the C2 region serves as a binding site for calcium. The C1 domain also acts as a target for tumor-promoting phorbol esters [4]. The nPKCs are similarly activated by DAG, phospholipids, and phorbol esters, but are not activated by calcium because of the lack of calcium-binding loops in the C2-like region [2]. The aPKCs have an atypical C1 domain and do not depend on DAG or calcium for activation [5, 6]. The activity of aPKCs is primarily regulated by protein-protein interactions through a Phox/Bem1 (PB1) domain located at the amino-terminus, which interacts with other

**Figure 1.** Schematic presentation of the PKC family. In mammals, the PKC family is composed of the following three structurally and functionally distinct subgroups: conventional PKCs (cPKCs; α, βI/II, and γ), novel PKCs (nPKCs; δ, ε, η, and θ), and atypical PKCs (aPKCs; ζ and ι/λ: λ in mice). aPKC (a regulator of epithelial cell polarity) lacks the proto-

Among the PKC family members, aPKCs play essential roles in establishing epithelial cell polarity by interacting with partition-defective (Par) proteins [9, 10]. The par genes were first identified in genetic screening for regulators of asymmetric division in the early embryo of *Caenorhabditis elegans* (*C. elegans*) [11, 12]. Par3 (a mammalian ortholog of *C. elegans* par-3) has been rediscovered as an aPKC-interacting protein [13]. Depletion of PKC-3 (an aPKC ortholog in *C. elegans*) using RNA interference results in a phenotype similar to that of par-3 and par-6 mutants [14, 15], leading to the discovery of physical and functional interactions among PKC-3, par-3, and par-6. Both Par3 and Par6 are PDZ domain-containing proteins and act as scaffold proteins. Indeed, Par6 and aPKCs form a stable heterodimer through their respective PB1 domains [7, 8]. Subsequently, Par6 serves as an adaptor protein for Rho family GTPases, Rac1, and Cdc42, in the activation of the Par6-aPKC heterodimer [16, 17]. The active form of Par6-aPKC, in turn, binds to Par3, which is mediated by the kinase domain of aPKCs and the

PB1 domain-containing proteins, such as PAR 6 (see below) [7, 8].

typic regulatory domain composed of C1 and C2 regions.

30 Hair and Scalp Disorders

Whole-body inactivation of aPKCλ results in embryonic lethality, which hampers further examination of the role of the aPKCλ-Par complex in epidermal homeostasis. To overcome this problem, two groups, including my group, generated mutant mice with epidermis-specific loss of aPKCλ using K5-Cre or K14-Cre mice [22, 23]. Although, in a strict sense, the distribution of K14-Cre transgene activity differs from that of K5-Cre transgene activity in the epidermis and hair follicle [25, 26], both mutant mice showed similar skin phenotypes. Thus, hereafter, when referring to findings common to both conditional knockout (cKO) mice, the term mutant mice or aPKCλ cKO mice is used, and when referring to findings obtained in the mutant mice associated with K5-Cre or K14-Cre individually, the term K5-cKO or K14-cKO mice is used, respectively.

Although aPKCλ cKO mice showed no gross anomalies at birth, they were easily distinguished from their control counterparts by the thinning of pledge hair from around postnatal day (P) 14 onward. The hair loss was progressive, and one-year-old mutant mice exhibited total alopecia (**Figure 2**) [22, 23]. The vibrissae were also shortened or were lost in the mutant mice [23].

K14-cKO mice showed impaired hair morphology. Scanning electron microscopy demonstrated that the regular cuticle pattern was lost in the mutant hair shafts. Consistent with this finding, the expressions of hair keratins in the inner root sheath (K28), cuticle (K35, K82, K85), cortex (K35, K81, K85), medulla (K28, K6, K75), and companion layer (K75, K6) were severely reduced [22].

**Figure 2.** Hair loss phenotype of mutant mice. Macroscopic presentation of a one-year-old control (left) and a K5-cKO mouse (right). Note that the vibrissae were lost in the mutant mouse (arrows).

### **3. Abnormal hair cycling**

During postnatal morphogenesis stages (P0–P16), the hair cycling of mutant hair follicles appeared to proceed normally, although the mutant integument became histologically noticeable with a thickened interfollicular epidermis (IFE) and enlarged sebaceous glands [22, 23]. However, entry into the first postnatal telogen (resting phase, normally starts around P18) was delayed in the mutant hair follicles. At P28, when control hair follicles entered into the first anagen (growth phase), they still had a long epithelial strand, a characteristic structure of catagen (regression phase) [27], and were positive for placental cadherin (P-cadherin), a marker for the epithelial strand. As a result, the start of the first anagen was delayed until P37 in the K5-cKO mice. In the K14-KO mice, the percentages of hair follicles that properly entered into catagen, telogen, and anagen were significantly reduced.

Moreover, mutant hair follicles were morphologically abnormal. They exhibited hyperkeratotic plugs and cyst-like structures with an expanded infundibulum and isthmus. Strikingly, these severely deformed mutant hair follicles regrew and entered into the second anagen much later than the control hair follicles. However, the mutant hair follicles in anagen did not proceed further into the second catagen or telogen. Instead, they started to degenerate, as revealed by the shrinking hair bulbs and reduced expressions of Ki67 and Lef1 [23]. In one-year-old mutant mice, the number of hair follicles was severely diminished.

Fibroblast growth factor 18 (Fgf18) is expressed in hair follicles and colocalized with keratin 15 (K15) and CD34 [28, 29], both of which are expressed in the bulge region at telogen. Fgf18 shows a cyclic expression pattern in hair follicles; its levels are low in anagen and high throughout telogen [29]. In mutant mice with epidermis-specific loss of Fgf18, the length of telogen was short, resulting in rapid succession of hair cycling [29]. Interestingly, the expression of Fgf18 was severely suppressed in the K5-cKO mice during hair morphogenesis and hair development [23]. Although precise molecular mechanisms associated with abnormal hair cycling in cKO mice remain to be elucidated, these results suggest that aPKCλ controls hair follicle cycling through Fgf18 signaling.
