Involvement of collagen XVII in pluripotency gene expression and metabolic reprogramming of lung cancer stem cells.

BACKGROUND: Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. METHODS: The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. RESULTS: We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3ß/ß-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. CONCULSIONS: These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.

Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation.

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Genome-wide approach identifies a novel gene-maternal pre-pregnancy BMI interaction on preterm birth.

Preterm birth (PTB) contributes significantly to infant mortality and morbidity with lifelong impact. Few robust genetic factors of PTB have been identified. Such 'missing heritability' may be partly due to gene × environment interactions (G × E), which is largely unexplored. Here we conduct genome-wide G × E analyses of PTB in 1,733 African-American women (698 mothers of PTB; 1,035 of term birth) from the Boston Birth Cohort. We show that maternal COL24A1 variants have a significant genome-wide interaction with maternal pre-pregnancy overweight/obesity on PTB risk, with rs11161721 (PG × E=1.8 × 10-8; empirical PG × E=1.2 × 10-8) as the top hit. This interaction is replicated in African-American mothers (PG × E=0.01) from an independent cohort and in meta-analysis (PG × E=3.6 × 10-9), but is not replicated in Caucasians. In adipose tissue, rs11161721 is significantly associated with altered COL24A1 expression. Our findings may provide new insight into the aetiology of PTB and improve our ability to predict and prevent PTB.

Matrix metalloproteinase (MMP)-19-deficient fibroblasts display a profibrotic phenotype.

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually lethal interstitial lung disease of unknown etiology characterized by aberrant activation of epithelial cells that induce the migration, proliferation and activation of fibroblasts. The resulting distinctive fibroblastic/myofibroblastic foci are responsible for the excessive extracellular matrix (ECM) production and abnormal lung remodeling. We have recently found that matrix metalloproteinase 19 (MMP-19)-deficient (Mmp19-/-) mice develop an exaggerated bleomycin-induced lung fibrosis, but the mechanisms are unclear. In this study, we explored the effect of MMP-19 deficiency on fibroblast gene expression and cell behavior. Microarray analysis of Mmp19-/- lung fibroblasts revealed the dysregulation of several profibrotic pathways, including ECM formation, migration, proliferation, and autophagy. Functional studies confirmed these findings. Compared with wild-type mice, Mmp19-/- lung fibroblasts showed increased α1 (I) collagen gene and collagen protein production at baseline and after transforming growth factor-ß treatment and increased smooth muscle-α actin expression (P < 0.05). Likewise, Mmp19-deficient lung fibroblasts showed a significant increase in proliferation (P < 0.01) and in transmigration and locomotion over Boyden chambers coated with type I collagen or with Matrigel (P < 0.05). These findings suggest that, in lung fibroblasts, MMP-19 has strong regulatory effects on the synthesis of key ECM components, on fibroblast to myofibroblast differentiation, and in migration and proliferation.

Molecular characterization of a nonfibrillar collagen from the marine sponge Chondrosia reniformis Nardo 1847 and positive effects of soluble silicates on its expression.

We report here the complete cDNA sequence of a nonfibrillar collagen (COLch) isolated from the marine sponge Chondrosia reniformis, Nardo 1847 using a PCR approach. COLch cDNA consists of 2,563 nucleotides and includes a 5' untranslated region (UTR) of 136 nucleotides, a 3' UTR of 198 nucleotides, and an open reading frame encoding for a protein of 743 amino acids with an estimated M (r) of 72.12 kDa. The phylogenetic analysis on the deduced amino acid sequence of C-terminal end shows that the isolated sequence belongs to the short-chain spongin-like collagen subfamily, a nonfibrillar group of invertebrate collagens similar to type IV collagen. In situ hybridization analysis shows higher expression of COLch mRNA in the cortical part than in the inner part of the sponge. Therefore, COLch seems to be involved in the formation of C. reniformis ectosome, where it could play a key role in the attachment to the rocky substrata and in the selective sediment incorporation typical of these organisms. qPCR analysis of COLch mRNA level, performed on C. reniformis tissue culture models (fragmorphs), also demonstrates that this matrix protein is directly involved in sponge healing processes and that soluble silicates positively regulate its expression. These findings confirm the essential role of silicon in the fibrogenesis process also in lower invertebrates, and they should give a tool for a sustainable production of marine collagen in sponge mariculture.

Differential induction of collagens by mechanical stress in human periodontal ligament cells.

OBJECTIVE: Excessive mechanical stress (MS) during hyperocclusion is known to result in destruction of periodontal tissues and alveolar bone, leading to occlusal trauma. Collagens are extracellular matrix components that are encoded by more than 30 different genes. They are classified into three types: fibril-forming, fibril-associated with interrupted triple helices (FACIT), and non-fibril forming collagens. Although MS is known to affect COL I, little is known about its effects on other types of collagens in the periodontal ligament (PDL). We hypothesised that MS could induce expression of the three different types of collagens, thus protecting against occlusal trauma. DESIGN: The aim of this study was to investigate intermittent uniaxial stretching-induced collagen expression in PDL cells using DNA microarray, polymerase chain reaction, and western blotting analysis. We compared changes in collagen expression caused by MS stimulation and osteogenic stimulation, and examined relationships between expression of collagen and their digestive enzymes, matrix metalloproteases (MMPs). RESULTS: Expression of both fibril-forming and FACIT collagens was transiently decreased in the initial phase after MS, while the expression of non-fibril-forming collagens was gradually increased. MS for 3-7 days resulted in gradual upregulation of all three types of collagen. Furthermore, the expression of fibril- and non-fibril-forming collagens was reciprocally related to expression of MMPs. In contrast, expression of all three types of collagen was slightly upregulated during osteogenesis. CONCLUSION: The MS-induced expression patterns of fibril-forming and FACIT collagens suggest changes in the composition of the extracellular matrix to increase the resistance of PDL cells to hyperocclusal force.

Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice.

Attempts to treat congenital protein deficiencies using bone marrow-derived cells have been reported. These efforts have been based on the concepts of stem cell plasticity. However, it is considered more difficult to restore structural proteins than to restore secretory enzymes. This study aims to clarify whether bone marrow transplantation (BMT) treatment can rescue epidermolysis bullosa (EB) caused by defects in keratinocyte structural proteins. BMT treatment of adult collagen XVII (Col17) knockout mice induced donor-derived keratinocytes and Col17 expression associated with the recovery of hemidesmosomal structure and better skin manifestations, as well improving the survival rate. Both hematopoietic and mesenchymal stem cells have the potential to produce Col17 in the BMT treatment model. Furthermore, human cord blood CD34(+) cells also differentiated into keratinocytes and expressed human skin component proteins in transplanted immunocompromised (NOD/SCID/gamma(c)(null)) mice. The current conventional BMT techniques have significant potential as a systemic therapeutic approach for the treatment of human EB.

Collagen XVII/BP180 protein expression in squamous cell carcinoma of the skin detected with novel monoclonal antibodies in archived tissues using tissue microarrays and digital microscopy.

Collagen XVII/BP180, a hemidesmosomal adhesion protein, is lost during normal keratinocyte maturation; however, it may be reexpressed upon malignant transformation. In this work, highly sensitive monoclonal antibodies 6D1 and 9G2 were produced, characterized, and used for the detection of collagen XVII in a tissue microarray series of archived samples of nonmelanocytic epithelial neoplasias, including 5 verruca vulgaris, 14 seborrheic keratosis, 38 actinic keratosis, 38 basal cell carcinoma (BCC), 15 basosquamous carcinoma, 58 squamous cell carcinoma (SCC), and 9 normal skin. Digital microscopy and a new tissue microarray software linking image and patient data allowed easy and validated evaluation and quality archiving of stained samples. In normal skin and benign epidermal lesions, collagen XVII protein was restricted to basal keratinocytes. However, possibly as a sign of undifferentiated/transformed state, it was widely expressed in SCC showing elevated levels around invasive tumor fronts with some staining in tumor adjacent stroma, endothelium, and histiocytes. Collagen XVII immunostaining of atypical keratinocytes in most actinic/solar keratosis supports the view of their malignancy and common origin with SCC. Squamous component of basosquamous carcinoma showed moderate reaction, whereas islets of BCC were mainly negative reflecting the diverse genotype and phenotype, and pathogenesis of SCC and BCC. These results suggest that collagen XVII neoexpression may be associated with early atypia/malignant transformation of keratinocytes. Further investigations are under way to analyze the potential of these antibodies for tracing progression and metastatic potential of skin tumors.

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.

Interferon regulatory factors (IRFs) are a family of transcriptional factors induced by interferon-gamma (IFN-gamma). 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-gamma, 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-gamma stimulation and possessed binding activity to IRF consensus sequence, showed preferential binding to the BPAG1-IRF sequence. Our results suggest that IFN-gamma-IRF system is involved in BPAG1 gene regulation in type-1 helper T-cell inflammatory skin conditions, such as psoriasis vulgaris.