Shuntaro Ikawa

p53 homologue, p51/p63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity.

p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of ΔNp51B (ΔNp63α), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, ΔNp51B by itself was found to have ability to induce expression of integrin α6β4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that ΔNp51B expression warrants integrin expression even under the influence of Notch1 and that ΔNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.

Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense.

Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms.

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.

p63/p51-induced Onset of Keratinocyte Differentiation via the c-Jun N-terminal Kinase Pathway Is Counteracted by Keratinocyte Growth Factor

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. ΔNp63α (ΔNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, ΔNp63α induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.

Ser46 phosphorylation of p53 is not always sufficient to induce apoptosis: multiple mechanisms of regulation of p53-dependent apoptosis

The tumor suppressor gene p53 plays a central role in determining cell fate in response to DNA damage; cells may undergo either senescence or apoptosis, depending on cell type. Phosphorylation of Serine 46 (Ser46) of p53 is considered to be a primary determinant for the induction of apoptosis, by selectively inducing transactivation of p53 target genes that have proapoptotic function. However, the generality of this mechanism of regulation of p53 remains a matter of debate. We investigated the role of p53 phosphorylation in adriamycin (ADR)‐induced apoptosis. We found that Ser46 was phosphorylated in four different cell lines undergoing ADR‐induced senescence, as well as in two different cell lines undergoing ADR‐induced apoptosis. Using alanine and glutamic acid substitution mutants of p53 Ser46, we showed that Ser46 phosphorylation is not a prerequisite for induction of the proapoptotic gene AIP1. These results indicate that Ser46 phosphorylation of p53 is not required for ADR‐induced apoptosis.

Identification of DRG family regulatory proteins (DFRPs): specific regulation of DRG1 and DRG2

DRG1 and DRG2 comprise a highly conserved subfamily of GTP‐binding proteins and are thought to act as critical regulators of cell growth. Their abnormal expressions may trigger cell transformation or cell cycle arrest. Our aim is to clarify their physiological functions and regulatory mechanisms. Here we report identification of novel proteins, DRG family regulatory protein (DFRP) 1 and DFRP2, which regulate expression of DRG proteins through specific binding. In transient transfection experiments, DFRP1 specifically binds DRG1, and DFRP2 preferentially binds DRG2. DFRPs provide stability to the target DRG proteins through physical association, possibly by blocking the poly‐ubiquitination that would precede proteolysis of DRG proteins. DFRPs are highly conserved in eucaryotes, and the expression patterns of dfrp1 and drg1 transcripts in Xenopus embryos and tissues are similar, indicating that these genes work cooperatively in various types of eukaryotic cells. Immunofluorescence experiments have revealed that the interaction between DRG1 and DFRP1 may occur in the cytoplasm. We generated dfrp1‐ knockout cells and found that endogenous expression of DRG1 is regulated by DFRP1, confirming that DFRP1 is a specific up‐regulator of DRG1 in vivo. On the basis of these results, we propose that DRG1 and DRG2 are regulated differently despite their structural similarities.

Bach1-dependent and -independent Regulation of Heme Oxygenase-1 in Keratinocytes

Bach1 is a member of the basic leucine zipper transcription factor family, and the Bach1/small Maf heterodimer specifically represses transcriptional activity directed by the Maf recognition element (MARE). Because Bach1 is a repressor of the oxidative stress response, we examined the function(s) of Bach1 in keratinocytes subjected to oxidative stress. Oxidative stress induced by H2O2 led to an increase in MARE activity and expression of heme oxygenase-1 (HO-1), an inducible antioxidant defense enzyme. Bach1 depletion by small interfering RNAs or by deletion of Bach1 enhanced HO-1 expression in the absence of H2O2, indicating that Bach1 is a critical repressor of HO-1 in keratinocytes. Although Bach1-deficient or -reduced keratinocytes expressed higher levels of HO-1 than control cells in response to H2O2, Bach1 down-regulation did not attenuate the production of reactive oxygen species by H2O2. In contrast, Bach1 overexpression abolished HO-1 induction by H2O2, which led to increased reactive oxygen species accumulation. HO-1 was induced during keratinocyte differentiation, but MARE activity did not change during differentiation. Furthermore, Bach1 overexpression did not inhibit differentiation-associated induction of HO-1 expression, suggesting that HO-1 induction in differentiation is independent of Bach1. Thus, in response to oxidative stress, Bach1 regulates the oxidation state through the negative control of HO-1 expression prior to terminal keratinocyte differentiation. However, Bach1-mediated repression is negated during keratinocyte differentiation.

p51/p63 inhibits ultraviolet B-induced apoptosis via Akt activation

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member ΔNp51B/ΔNp63α (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, ΔNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of ΔNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that ΔNp51B blocks keratinocyte apoptosis. Although the exogenous expression of ΔNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH2-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate.

p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53‐responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti‐cancer therapy.

Reversible switching of expression of c-kit and Pax-5 in immature hematopoietic progenitor cells by stromal cells.

OBJECTIVEnBone marrow stromal cells provide the microenvironment for self-renewal and differentiation of hematopoietic stem/progenitor cells through complex cell-cell interaction. To elucidate the regulatory mechanisms of hematopoiesis by stromal cells, we established a novel stroma-dependent hematopoietic cell line and explored the phenotypic changes regulated by the two stromal cells.nnnMATERIALS AND METHODSnDFC-28 cells clonally established from long-term bone marrow culture of C57BL/6 mice were sustained by coculture on MSS62 cells (mouse spleen stromal cell line). When DFC-28 cells were transferred to TBR31-1 cells (mouse bone marrow stromal cell line), their phenotypic changes were analyzed by flow cytometry and reverse transcriptase polymerase chain reaction.nnnRESULTSnDFC-28 cells on MSS62 cells exhibited surface phenotypes of the immature hematopoietic progenitor cells (Lin(-)AA4.1(+)c-kit(+)Sca-1(-)). By stroma-replacement from MSS62 cells to TBR31-1 cells, DFC-28 cells were differentiated into very early B-lymphoid stage characterized by c-kit down-regulation and induction of BP-1 and B-lymphoid-associated genes (Pax-5, CD19, TdT, Rag-1, and Rag-2). In addition, the differentiation phenotypes reverted to the immature state characterized by c-kit induction and down-regulation of BP-1 and B-lymphoid-associated genes by replacing stroma back to MSS62 from TBR31-1. Interleukin-7 stimulation and conditioned medium of TBR31-1 cells were ineffective in converting the differentiation phenotypes of DFC-28 cells.nnnCONCLUSIONSnThe results demonstrate that the differentiation phenotypes and growth potential of stroma-dependent hematopoietic progenitor cells we established could be reversibly controlled via direct contact with stromal cells in the microenvironment.