Masashi Miyai

MafB interacts with Gcm2 and regulates parathyroid hormone expression and parathyroid development.

Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid‐thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v‐maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB+/− mice were mislocalized between the thymus and thyroid. In mafB−/− mice, the parathyroid did not separate from the thymus. Furthermore, in mafB−/− mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation.

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

Background: Caspase-14 activation is associated with keratinocyte terminal differentiation. Results: Kallikrein-related peptidase-7 (KLK7) generates an activation intermediate comprising 20- and 8-kDa subunits by cleavage after Tyr178 of procaspase-14. This intermediate cleaves procaspase-14 at Asp146, generating mature, active caspase-14. Conclusion: Caspase-14 maturation is mediated by KLK7 and an intermediate form of caspase-14. Significance: Caspase-14 has a unique activation mechanism. The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr178, generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. Km values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μm, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.

Mesotrypsin and caspase-14 participate in prosaposin processing: potential relevance to epidermal permeability barrier formation.

Background: The mechanism of prosaposin processing to generate saposins A-D is unknown. Results: Epidermis-specific mesotrypsin and caspase-14 generated mature saposins from prosaposin. Deficiency of prosaposin or saposin A resulted in loss of intercellular lipid components necessary for maintenance of skin permeability barrier properties. Conclusion: Mesotrypsin and caspase-14 are involved in prosaposin processing. Significance: Saposin generation in epidermis is regulated in a differentiation-associated manner. A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A–D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A–D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.

c-Maf and MafB transcription factors are differentially expressed in Huxley's and Henle's layers of the inner root sheath of the hair follicle and regulate cuticle formation

BACKGROUND The hair follicle of mammalian skin consists of a group of concentric epithelial cell layers. The inner root sheath (IRS), which surrounds the hardening hair shaft beneath the skin surface, is subdivided into three layers, termed the cuticle of the IRS, Huxleys layer, and Henles layer. The IRS forms a follicular wall in the hair canal and helps guide the developing hair shaft. c-Maf and MafB, members of the Maf family of transcription factors, play important roles in the developmental processes of various tissues and in cell type-specific gene expression. OBJECTIVE The aim of this study is to reveal the pattern of expression and functional roles of c-Maf and MafB in the hair follicle. METHODS We determined the precise location of c-Maf and MafB expression using immunofluorescent staining of mouse skin sections with layer-specific markers. We also analyzed whiskers of c-maf- and mafB-null mice (c-maf(-/-) and mafB(-/-), respectively) using scanning electron microscopy. RESULTS c-Maf and MafB were differentially expressed in the Huxleys and Henles layers of the IRS. Scanning electron microscopic analysis showed irregular cuticle patterning of whiskers of c-maf(-/-) and mafB(-/-) mice. The cuticles of mafB(-/-) mice were also thinner than those of wild-type mice. CONCLUSION c-Maf and MafB are expressed in the IRS layers in a lineage-restricted manner and are involved in hair morphogenesis.

Identification of an S100A8 Receptor Neuroplastin-β and its Heterodimer Formation with EMMPRIN

We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-β as an unreported S100A8 receptor. Neuroplastin-β and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-β recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-β and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis.

Key component of inflammasome, NLRC4, was identified in the lesional epidermis of psoriatic patients

Inflammasomes are multimolecular complexes that control the inflammatory response. The function of inflammasomes in the pathogenesis of psoriasis is still unclear. To clarify the relationship between inflammasomes and the pathophysiology of psoriasis, and in particular, to identify molecules interacting with caspase‐1, a crucial component of inflammasomes, scale extracts obtained from patients with psoriasis were immunoprecipitated with anti‐caspase‐1 antibody and analyzed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC‐MS/MS). The expression of the inflammasome component was assessed by immunohistochemical analysis and an in vitro assay. We identified several candidates for caspase‐1‐interacting proteins from the psoriatic scale extracts by immunoprecipitation and LC‐MS/MS. Nucleotide‐binding oligomerization domain‐containing protein‐like receptor family CARD domain‐containing protein 4 (NLRC4) was the only inflammasome component among the candidates; thus, the protein is considered to be a key factor of inflammasomes in psoriasis. No inflammasome component was found in the extracts of atopic dermatitis or normal skin by LC‐MS/MS. Immunohistochemical analysis demonstrated upregulation of NLRC4 in the lesional epidermis of some psoriatic patients whereas weak expression of NLRC4 was detected in the normal and non‐lesional epidermis. The mRNA expression of the NLRC4 gene increased in keratinocytes at confluency, 48 h after air exposure and after the addition of 1.5 mmol/L calcium chloride. Our findings suggest that NLRC4 may be involved in the exacerbation or modification of psoriatic lesions.

Ectopic expression of the transcription factor MafB in basal keratinocytes induces hyperproliferation and perturbs epidermal homeostasis

Mammalian epidermis is composed of four morphologically and functionally distinct layers of keratinocytes. The innermost basal layer consists of proliferating self‐renewing keratinocytes, which also undergo asymmetric cell division to differentiate into postmitotic suprabasal cells throughout life. Control of the balance between growth and differentiation of basal cells is important for epidermal homeostasis to prevent skin disorders including malignancies; however, the underlying mechanism remains to be elucidated. Recently, MafB was identified as one of the transcription factors that regulate epidermal keratinocyte differentiation. MafB is expressed in postmitotic differentiating keratinocytes, and epidermal differentiation is partially impaired in MafB‐deficient mice. To further establish the roles of MafB in the epidermis in vivo, we generated mice transgenic for MafB under the control of the basal cell‐specific keratin (Krt) 14 promoter. In the epidermis of transgenic mice at embryonic day 18.5, the number of proliferating Krt14‐positive basal‐like cells was increased, and the granular and cornified layers were thickened. Furthermore, these MafB transgenic mice developed papillomas spontaneously with age. Therefore, MafB promotes differentiation in postmitotic keratinocytes and simultaneously has potential to promote growth when ectopically expressed in undifferentiated basal keratinocytes.