Takehiko Yamazaki

Circulating bone marrow-derived osteoblast progenitor cells are recruited to the bone-forming site by the CXCR4/stromal cell-derived factor-1 pathway.

Previous studies demonstrated the existence of osteoblastic cells in circulating blood. Recently, we reported that osteoblast progenitor cells (OPCs) in circulation originated from bone marrow and contributed to the formation of ectopic bone induced by implantation of a bone morphogenetic protein (BMP)‐2‐containing collagen pellet in mouse muscular tissue. However, the character of circulating bone marrow‐derived osteoblast progenitor cells (MOPCs) and the precise mechanisms involving the circulating MOPCs in the osteogenic processes, such as signals that recruit the circulating MOPCs to the osseous tissues, have been obscure. In this report, we demonstrated for the first time that the MOPCs were mobilized from intact bones to transiently occupy approximately 80% of the mononuclear cell population in the circulating blood by BMP‐2‐pellet implantation. The mobilized MOPCs in the circulation did not express the hematopoietic marker CD45 on their surface, but they expressed CD44 and CXCR4, receptors of osteopontin and stromal cell‐derived factor‐1 (SDF‐1), respectively. The MOPCs isolated from the mouse peripheral blood showed the ability to be osteoblasts in vitro and in vivo. Furthermore, the MOPCs in the circulation efficiently migrated to the region of bone formation by chemoattraction of SDF‐1 expressed in vascular endothelial cells and the de novo osteoblasts of the region. These data may provide a novel insight into the mechanism of bone formation involving MOPCs in circulating blood, as well as perspective on the use of circulating MOPCs to accelerate bone regeneration in the future.

PDGFRα-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia

The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin−/PDGFRα+) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin−/PDGFRα+ cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.

Bone Marrow Cell Transfer into Fetal Circulation Can Ameliorate Genetic Skin Diseases by Providing Fibroblasts to the Skin and Inducing Immune Tolerance

Recent studies have shown that skin injury recruits bone marrow-derived fibroblasts (BMDFs) to the site of injury to accelerate tissue repair. However, whether uninjured skin can recruit BMDFs to maintain skin homeostasis remains uncertain. Here, we investigated the appearance of BMDFs in normal mouse skin after embryonic bone marrow cell transplantation (E-BMT) with green fluorescent protein-transgenic bone marrow cells (GFP-BMCs) via the vitelline vein, which traverses the uterine wall and is connected to the fetal circulation. At 12 weeks of age, mice treated with E-BMT were observed to have successful engraftment of GFP-BMCs in hematopoietic tissues accompanied by induction of immune tolerance against GFP. We then investigated BMDFs in the skin of the same mice without prior injury and found that a significant number of BMDFs, which generate matrix proteins both in vitro and in vivo, were recruited and maintained after birth. Next, we performed E-BMT in a dystrophic epidermolysis bullosa mouse model (col7a1(-/-)) lacking type VII collagen in the cutaneous basement membrane zone. E-BMT significantly ameliorated the severity of the dystrophic epidermolysis bullosa phenotype in neonatal mice. Type VII collagen was deposited primarily in the follicular basement membrane zone in the vicinity of the BMDFs. Thus, gene therapy using E-BMT into the fetal circulation may offer a potential treatment option to ameliorate genetic skin diseases that are characterized by fibroblast dysfunction through the introduction of immune-tolerated BMDFs.

Serum Anti-BPAG1 Auto-Antibody Is a Novel Marker for Human Melanoma

Malignant melanoma is one of the most aggressive types of tumor. Because malignant melanoma is difficult to treat once it has metastasized, early detection and treatment are essential. The search for reliable biomarkers of early-stage melanoma, therefore, has received much attention. By using a novel method of screening tumor antigens and their auto-antibodies, we identified bullous pemphigoid antigen 1 (BPAG1) as a melanoma antigen recognized by its auto-antibody. BPAG1 is an auto-antigen in the skin disease bullous pemphigoid (BP) and anti-BPAG1 auto-antibodies are detectable in sera from BP patients and are used for BP diagnosis. However, BPAG1 has been viewed as predominantly a keratinocyte-associated protein and a relationship between BPAG1 expression and melanoma has not been previously reported. In the present study, we show that bpag1 is expressed in the mouse F10 melanoma cell line in vitro and F10 melanoma tumors in vivo and that BPAG1 is expressed in human melanoma cell lines (A375 and G361) and normal human melanocytes. Moreover, the levels of anti-BPAG1 auto-antibodies in the sera of melanoma patients were significantly higher than in the sera of healthy volunteers (p<0.01). Furthermore, anti-BPAG1 auto-antibodies were detected in melanoma patients at both early and advanced stages of disease. Here, we report anti-BPAG1 auto-antibodies as a promising marker for the diagnosis of melanoma, and we discuss the significance of the detection of such auto-antibodies in cancer biology and patients.

Systemic Administration of a Novel Immune-Stimulatory Pseudovirion Suppresses Lung Metastatic Melanoma by Regionally Enhancing IFN-γ Production

Purpose: Cancer immunotherapy has encountered many difficulties in the face of the expectation to eradicate cancer, and new breakthroughs are required. We have previously shown that UV-inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) induce immunity against multiple tumor types. In this study, a novel pseudovirion that stimulates more robust antitumor immunity was designed for cancer treatment. Experimental Design: First, we found that culturing murine splenocytes with HVJ-E in combination with interleukin (IL)-12 resulted in a remarkable increase in IFN-γ production compared with that observed in splenocytes cultured with IL-12 alone. The synergistic effects of HVJ-E and IL-12 on IFN-γ production were caused by viral F proteins independently of HVJ-E fusion activity and not by hemagglutination from hemagglutinin-neuraminidase (HN) proteins. We next constructed HN-depleted HVJ-E expressing the Fc region of immunoglobulin G (IgG) on the envelope and single-chain IL-12 containing the ZZ domain of protein A to produce an IL-12–conjugated HVJ-E particle without hemagglutinating activity. Results: IL-12–conjugated HVJ-E dramatically enhanced the amount of IFN-γ produced by immune cells. Intratumoral injection of IL-12–conjugated HVJ-E eradicated murine melanomas more effectively than injection of wild-type HVJ-E through increased production of melanoma-specific CTLs. IL-12–conjugated HVJ-E preferentially accumulated in the lungs after systemic administration. When small metastatic melanoma foci were formed in the lungs, systemic administration of IL-12–conjugated HVJ-E significantly reduced the number of metastatic foci by inducing local production of IFN-γ in the lungs and generating large numbers of melanoma-specific CTLs. Conclusion: IL-12–conjugated HVJ-E is a promising tool for the treatment of cancers, including lung metastasis. Clin Cancer Res; 19(3); 668–79. ©2012 AACR.

Enhancement of ultraviolet-induced apoptosis by NF-κB decoy oligonucleotides

Background  A decoy strategy utilizing oligonucleotides (ODN) containing the specific binding sequence of a certain transcription factor has been developed and is considered to be a potential new class of antigene therapy. However, the application of this new therapeutic modality to skin diseases has not been fully documented.

Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS.

Abstract:  The 230‐kDa bullous pemphigoid antigen (BPAG1) is an integral component of hemidesmosomes. We have previously reported that interferon‐γ (IFNγ) inhibits the transcription of the BPAG1 gene ( 1 ). Here we investigated the target sequences of IFNγ‐signal transduction pathway in the BPAG1 promoter in epidermal keratinocytes. Transient transfections with 5′‐deletion constructs of BPAG1 promoter‐luciferase reporter gene plasmids in cultured normal human epidermal keratinocytes (NHEK) allowed us to narrow the DNA region containing IFNγ inhibitory element (IGIE) to between −1 and −89, upstream from the transcription initiation site (+1). Homology search in this region identified a chimeric sequence, consisting of IFN‐stimulated responsive element (ISRE) with a partial 7‐bp sequence of IFNγ activation site (GAS), as identified in the guanylate‐binding protein (GBP) gene, inserted at its center. Functional analysis of IGIE, inserted in front of the heterologous thymidine kinase promoter, indicated that IGIE acts as a down‐regulatory element of the promoter through IFNγ‐dependent signal pathway. Transient transfection studies with BPAG1 promoter‐reporter gene constructs containing mutated IGIE (with TT to GG transversions in the region of 5′ISRE, GAS, and 3′ISRE) demonstrated that disruption of the ISRE sequences, but not GAS, markedly suppressed the BPAG1 basal promoter activity and resulted in attenuated IFNγ response in keratinocytes. Our findings provide novel insight into the mechanism of IFNγ regulation in keratinocyte differentiation and proliferation.

Transcriptional regulation of the 230‐kDa bullous pemphigoid antigen gene expression by interferon regulatory factor 1 and interferon regulatory factor 2 in normal human epidermal keratinocytes

Abstract:  Interferon regulatory factors (IRFs) are a family of transcriptional factors induced by interferon‐γ (IFN‐γ). Recent studies have indicated that the deregulation of IRF system in keratinocytes is responsible, at least in part, for aberrant proliferation and the differentiation of the psoriatic epidermis. Previously, we reported that the expression of 230‐kDa bullous pemphigoid antigen (BPAG1) gene, which is strictly restricted to basal keratinocytes, is transcriptionally suppressed by IFN‐γ, but the contribution of IRFs in such suppression is still unclear. In this study, we investigated the role of IRFs in the regulation of BPAG1 gene expression. Computer analysis identified IRF1 and IRF2 consensus sequences between −135 and −123 on BPAG1 promoter region. Transient transfection studies with BPAG1 promoter‐luciferase reporter gene plasmids and IRF1 and IRF2 expression plasmids revealed that IRF1 and IRF2 directly down‐regulated BPAG1 gene transcription in cultured normal human epidermal keratinocytes. Several sets of gel retardation assays with the BPAG1–IRF binding sequence as a probe indicated that IRF1 and IRF2 could bind to the BPAG1–IRF sequence, but some other protein(s), which was induced by IFN‐γ stimulation and possessed binding activity to IRF consensus sequence, showed preferential binding to the BPAG1–IRF sequence. Our results suggest that IFN‐γ–IRF system is involved in BPAG1 gene regulation in type‐1 helper T‐cell inflammatory skin conditions, such as psoriasis vulgaris.

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain

Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k(1) (0.19 min(-1))>>k(2) (0.0008 min(-1)) approximately k(3) (0.0006 min(-1)) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k(cat) and kcat/Km values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.

Polymorphic glutathione S-transferase subunit 3 of rat liver exhibits different susceptibilities to carbon tetrachloride: differences in their interactions with heat-shock protein 90.

Rat glutathione S-transferase (GST) subunit 3 gene has polymorphism, one type encoding Asn(198)-Cys(199) (NC type) and another encoding Lys(198)-Ser(199) (KS type). To examine whether the two types of GST 3-3 exhibit different susceptibilities to oxidative stress in vivo, rats were administered with CCl(4), a hepatotoxin causing severe oxidative stress, and its effect on liver GST 3-3 was compared. Decrease in GST activities in liver due to CCl(4) administration was more evident in NC type rats than in KS type rats, and most GST activities of KS type rats were confined to S-hexylglutathione-Sepharose, whereas those of NC type rats were not. Decreases in GST subunits 1 and 3 were more marked in NC type rats and glutathiolated NC type GST 3-3 was also detected. These results indicated that KS and NC type GST 3-3 of rat livers exhibited different susceptibilities to CCl(4) in vivo. A protein consisting of a subunit with molecular mass of 90 kDa was shown to bind to KS type GST 3-3 but not to NC type. This protein was identified as heat-shock protein (HSP) 90beta by N-terminal amino acid sequencing and immunoblotting. A specific HSP90 inhibitor geldanamycin released their binding. There was no difference in the binding of apoptosis signal-regulating kinase 1 to GST 3-3 between NC and KS type rats. These findings suggest that HSP90 interacts with KS type GST 3-3 and thereby protects it from inactivation due to CCl(4).