Epitranscriptomics of Ischemic Heart Disease-The IHD-EPITRAN Study Design and Objectives.

Epitranscriptomic modifications in RNA can dramatically alter the way our genetic code is deciphered. Cells utilize these modifications not only to maintain physiological processes, but also to respond to extracellular cues and various stressors. Most often, adenosine residues in RNA are targeted, and result in modifications including methylation and deamination. Such modified residues as N-6-methyl-adenosine (m6A) and inosine, respectively, have been associated with cardiovascular diseases, and contribute to disease pathologies. The Ischemic Heart Disease Epitranscriptomics and Biomarkers (IHD-EPITRAN) study aims to provide a more comprehensive understanding to their nature and role in cardiovascular pathology. The study hypothesis is that pathological features of IHD are mirrored in the blood epitranscriptome. The IHD-EPITRAN study focuses on m6A and A-to-I modifications of RNA. Patients are recruited from four cohorts: (I) patients with IHD and myocardial infarction undergoing urgent revascularization; (II) patients with stable IHD undergoing coronary artery bypass grafting; (III) controls without coronary obstructions undergoing valve replacement due to aortic stenosis and (IV) controls with healthy coronaries verified by computed tomography. The abundance and distribution of m6A and A-to-I modifications in blood RNA are charted by quantitative and qualitative methods. Selected other modified nucleosides as well as IHD candidate protein and metabolic biomarkers are measured for reference. The results of the IHD-EPITRAN study can be expected to enable identification of epitranscriptomic IHD biomarker candidates and potential drug targets.

Novel DSP Spectrin 6 Region Variant Causes Neonatal Erythroderma, Failure to Thrive, Severe Herpes Simplex Infections and Brain Lesions.

Desmoplakin (DSP) and Desmoglein 1 (DSG1) variants result in skin barrier defects leading to erythroderma, palmoplantar keratoderma and variable [AQ4] other features. Some DSG1 variant carriers present with SAM syndrome (Severe dermatitis, multiple Allergies, Metabolic wasting) and a SAM-like phenotype has been reported in 4 subjects with different heterozygous DSP variants. We report here a patient with a novel DSP spectrin region (SR) 6 variant c.1756C>T, p.(His586Tyr), novel features of brain lesions and severe recurrent mucocutaneous herpes simplex virus infections, with a favourable response to ustekinumab. Through a review of reported cases of heterozygous variants in DSP SR6 (n = 15) and homozygous or compound heterozygous variants in DSG1 (n = 12) and SAM-like phenotype, we highlight phenotypic variability. Woolly hair, nail abnormalities and cardiomyopathy characterize patients with DSP variants, while elevated immunoglobulin E and food allergies are frequent in patients with DSG1 variants. Clinicians should be aware of the diverse manifestations of desmosomopathies.

Intracellular signalling pathways and cytoskeletal functions converge on the psoriasis candidate gene CCHCR1 expressed at P-bodies and centrosomes.

BACKGROUND: CCHCR1 (Coiled-Coil α-Helical Rod protein 1) is a putative psoriasis candidate gene with the risk alleles CCHCR1*WWCC and *Iso3, the latter inhibiting the translation of isoform 1. CCHCR1 was recently shown to be a centrosomal protein, as well as a component of cytoplasmic processing bodies (P-bodies) that regulate mRNA turnover. The function of CCHCR1 has remained unsettled, partly because of the inconsistent findings; it has been shown to play a wide variety of roles in divergent processes, e.g., cell proliferation and steroidogenesis. Here we utilized RNA sequencing (RNAseq) using HEK293 cells overexpressing isoforms 1 or 3 (Iso1, Iso3 cells), in combination with the coding non-risk or risk (*WWCC) haplotype of CCHCR1. Our aim was to study the overall role of CCHCR1 and the effects of its variants. RESULTS: The overexpression of CCHCR1 variants in HEK293 cells resulted in cell line-specific expression profiles though several similarities were observable. Overall the Iso1 and Iso3 cells showed a clear isoform-specific clustering as two separate groups, and the Non-risk and Risk cells often exhibited opposite effects. The RNAseq supported a role for CCHCR1 in the centrosomes and P-bodies; the most highlighted pathways included regulation of cytoskeleton, adherens and tight junctions, mRNA surveillance and RNA transport. Interestingly, both the RNAseq and immunofluorescent localization revealed variant-specific differences for CCHCR1 within the P-bodies. CONCLUSIONS: CCHCR1 influenced a wide variety of signaling pathways, which could reflect its active role in the P-bodies and centrosomes that both are linked to the cytoskeleton; as a centrosomal P-body protein CCHCR1 may regulate diverse cytoskeleton-mediated functions, such as cell adhesion and -division. The present findings may explain the previous inconsistent observations about the functions of CCHCR1.

Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control.

BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice.

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.

Discovery of increased epidermal DNAH10 expression after regeneration of dermis in a randomized with-in person trial - reflections on psoriatic inflammation.

Because molecular memories of past inflammatory events can persist in epidermal cells, we evaluated the long-term epidermal protein expression landscapes after dermal regeneration and in psoriatic inflammation. We first characterized the effects of two dermal regeneration strategies on transplants of indicator split-thickness skin grafts (STSGs) in ten adult patients with deep burns covering more than 20% of their body surface area. After fascial excision, three adjacent areas within the wound were randomized to receive a permanent dermal matrix, a temporary granulation-tissue-inducing dressing or no dermal component as control. Control areas were covered with STSG immediately, and treated areas after two-weeks of dermis formation. Epidermis-dermis-targeted proteomics of one-year-follow-up samples were performed for protein expression profiling. Epidermal expression of axonemal dynein heavy chain 10 (DNAH10) was increased 20-fold in samples having had regenerating dermis vs control. Given the dermal inflammatory component found in our dermal regeneration samples as well as in early psoriatic lesions, we hypothesized that DNAH10 protein expression also would be affected in psoriatic skin samples. We discovered increased DNAH10 expression in inflammatory lesions when compared to unaffected skin. Our results associate DNAH10 expression with cell proliferation and inflammation as well as with the epidermal memory resulting from the previous regenerative signals of dermis. This study (ISRCTN14499986) was funded by the Finnish Ministry of Defense and by government subsidies for medical research.

Somatic FGF9 mutations in colorectal and endometrial carcinomas associated with membranous beta-catenin.

We previously described striking molecular features including high frequency of membranous beta-catenin in subsets of familial colon cancers with as yet unknown predisposition. We hypothesized that such tumors might carry mutations in Wnt/beta-catenin target genes. Fibroblast growth factor 9 (FGF9) was an attractive target, as it maps to a common area of loss of heterozygosity (LOH) in colorectal carcinomas on 13q12.11. Here, we report, for the first time, the occurrence of FGF9 mutations in human cancers. We found a total of six distinct FGF9 mutations including one frameshift, four missense, and one nonsense, in 10 (six colorectal and four endometrial) out of 203 tumors and cell lines. The frameshift mutation was detected in five different tumors. Mapping of these mutations onto the crystal structure of FGF9 predicted that they should all lead to loss of function albeit through variable mechanisms. The p.R173K mutation should diminish ligand affinity for heparin/heparan sulfate, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations should negatively impact ligand's interaction with receptor, while p.G84E and p.E142X (FGF9(Delta142-208)) mutations should interfere with ligand folding. Consistent with these structural predictions, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations impaired the ability of ligand to activate mitogen-activated protein kinase (MAPK) cascade in cultured cells expressing FGF receptors. LOH was observed in seven out of nine FGF9 mutant tumors, supporting the predicted loss of function. Interestingly, eight out of 10 (80%) of the FGF9 mutant tumors showed normal membranous beta-catenin expression and the absence of mutation in the beta-catenin gene (CTNNB1). These data suggest that FGF9 plays a role in colorectal and endometrial carcinogenesis.

The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes.

The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.

A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR.

Chloride absorption and bicarbonate excretion through exchange by the solute carrier family 26 member 3 (SLC26A3) and cystic fibrosis transmembrane conductance regulator (CFTR) are crucial for many tissues including sperm and epithelia of the male reproductive tract. Homozygous SLC26A3 mutations cause congenital chloride diarrhea with male subfertility, while homozygous CFTR mutations cause cystic fibrosis with male infertility. Some homozygous or heterozygous CFTR mutations only manifest as male infertility. Accordingly, we studied the influence of SLC26A3 on idiopathic infertility by sequencing exons of SLC26A3 in 283 infertile and 211 control men. A heterozygous mutation c.2062 G > C (p.Asp688His) appeared in nine (3.2%) infertile men, and additionally, in two (0.9%) control men, whose samples revealed a sperm motility defect. The p.Asp688His mutation is localized in the CFTR-interacting STAS domain of SLC26A3 and enriched in Finland, showing a significant association with male infertility in comparison with 6,572 Finnish (P < 0.05) and over 120,000 global alleles (P < 0.0001) (ExAC database). Functional studies showed that while SLC26A3 is a strong activator of CFTR-dependent anion transport, SLC26A3-p.Asp688His mutant retains normal Cl-/HCO3- exchange activity but suppresses CFTR, despite unaffected domain binding and expression. These results suggest a novel mechanism for human male infertility─impaired anion transport by the coupled SLC26A3 and CFTR.

NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis.

Psoriatic skin differs distinctly from normal skin by its thickened epidermis. Most gene expression comparisons utilize full-thickness biopsies, with substantial amount of dermis. We assayed the transcriptomes of normal, lesional, and non-lesional psoriatic epidermis, sampled as split-thickness skin grafts, with 5'-end RNA sequencing. We found that psoriatic epidermis contains more mRNA per total RNA than controls, and took this into account in the bioinformatic analysis. The approach highlighted innate immunity-related pathways in psoriasis, including NOD-like receptor (NLR) signaling and inflammasome activation. We demonstrated that the NLR signaling genes NOD2, PYCARD, CARD6, and IFI16 are upregulated in psoriatic epidermis, and strengthened these findings by protein expression. Interestingly, PYCARD, the key component of the inflammasome, showed an altered expression pattern in the lesional epidermis. The profiling of non-lesional skin highlighted PSORS4 and mitochondrially encoded transcripts, suggesting that their gene expression is altered already before the development of lesions. Our data suggest that all components needed for the active inflammasome are present in the keratinocytes of psoriatic skin. The characterization of inflammasome pathways provides further opportunities for therapy. Complementing previous transcriptome studies, our approach gives deeper insight into the gene regulation in psoriatic epidermis.

BMPR2 mutations have short lifetime expectancy in primary pulmonary hypertension.

In a nationwide study, we identified a total of 59 patients diagnosed with primary pulmonary hypertension (PPH) in Finland between the years 1987 and 1999. These data support a minimum estimate for a PPH population prevalence of 5.8 cases/million with an incidence of 0.2-1.3 cases/million/year. The male-to-female ratio among the patients was 1:4, while 7% (4/59) of the PPH probands had a known family history of the disorder. Familial or sporadic PPH showed no geographic clustering to any region of Finland. Sequencing of the coding regions and exon-intron boundaries of the bone morphogenetic protein receptor type 2 (BMPR2) identified heterozygous BMPR2 mutations in 12% (3/26) of the sporadic and 33% (1/3) of the familial patients. All four mutations were different, and two of those have been previously reported in other populations. Pathogenic defects in BMPR2 include a novel missense mutation (c.2696G>C encoding R899P), located within the receptor intracellular cytoplasmic domain whose function has been poorly characterized. Our analysis demonstrates that this mutant, while localizing to the cell surface, does not impact on SMAD-mediated (mothers against decapentaplegic homolog) intracellular signaling, but leads to constitutive activation of the p38(MAPK) pathway. The absence of a founder mutation in a genetically homogeneous population, such as the Finns, suggests that all identified BMPR2 mutations have to be rather young while the ancestral (if any) mutations have been lost either due to repetitive genetic bottlenecks or due to significant negative selection. Hum Mutat 26(2), 1-6, 2005. (c) 2005 Wiley-Liss, Inc.

Healing a natural knockout of epithelial organogenesis.

Ectodermal dysplasias are a large group of rare genetic disorders with developmental abnormalities in skin, teeth, hair and nails. Many of them are clinically serious and impair the life of patients. The cloning of the gene for the most common of them, X-linked anhidrotic ectodermal dysplasia, in 1996 opened the door to dissect novel developmental pathways at the molecular level. Since then, several new genes and proteins with novel functions have been identified.

SLC26A6 and SLC26A7 anion exchangers have a distinct distribution in human kidney.

BACKGROUND: The anion transporters SLC26A6 (PAT1) and SLC26A7, transporting at least chloride, oxalate, sulfate and bicarbonate, show a distinct expression and function in different mammalian species. They are expressed in kidney, but their exact localization in human kidney has not been studied. We therefore examined SLC26A6 and A7 expression in human kidneys. METHODS: The localization of SLC26A6 and A7 in different segments of human nephrons was studied by RT-PCR and immunohistochemistry by comparing to the tubular markers PNRA, CD10, Tamm-Horsfall antigen, high molecular weight cytokeratin, CK7, AQP2 and H(+)V-ATPase. RESULTS: In human kidney, SLC26A6 is expressed in distal segments of proximal tubules, parts of the thin and thick ascending limbs of Henle's loops, macula densa, distal convoluted tubules and a subpopulation of intercalated cells of collecting ducts. SLC26A7 is expressed in extraglomerular mesangial cells and a subpopulation of intercalated cells of collecting ducts. CONCLUSION: Our results show that in human kidney SLC26A6 and A7 have a distinct, partially overlapping expression in distal segments of nephrons. The distribution partly differs from that found previously in rodent kidneys.

HCR, a candidate gene for psoriasis, is expressed differently in psoriasis and other hyperproliferative skin disorders and is downregulated by interferon-gamma in keratinocytes.

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.

Matrix metalloproteinase-21, the human orthologue for XMMP, is expressed during fetal development and in cancer.

We have characterized a novel human matrix metalloproteinase (MMP-21) from human placenta DNA complementary to RNA (cDNA). The 569 amino acid translation of the cDNA includes all the typical features of an MMP family member, namely a signal sequence, a prodomain with a PRCGVPD motif, a zinc-binding catalytic domain with an HEIGHVLGL sequence, and a hemopexin-like domain flanked by two cysteine residues. Furthermore, MMP-21 has a furin activation sequence, but no transmembrane sequence nor a cytoplasmic domain. As in Xenopus laevis and Cynops pyrrhogaster there is an additional insertion of approximately 30 amino acids between the prodomain and the catalytic domain, which is poorly conserved between the species and is in human MMP-21 especially proline rich. The MMP-21 gene has seven exons and is located in chromosome 10. This new MMP is the human orthologue for XMMP and CyMMP expressed during gastrulation of X. laevis and C. pyrrhogaster, respectively. A 2.5 kb messenger RNA was observed in fetal liver by Northern analysis. By reverse transcription-polymerase chain reaction, MMP-21 is expressed in various human fetal and adult tissues as well as in cancer cell lines. MMP-21 protein can also be detected in malignancies such as ovarian and colon carcinomas by immunohistochemical staining. Our findings suggest that MMP-21 functions in embryogenesis and tumor progression.

Centrosomal localization of the psoriasis candidate gene product, CCHCR1, supports a role in cytoskeletal organization.

CCHCR1 (Coiled-Coil α-Helical Rod protein 1), within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene with the psoriasis associated risk allele CCHCR1*WWCC. Although its expression pattern in psoriatic skin differs from healthy skin and its overexpression influences cell proliferation in transgenic mice, its role as a psoriasis effector gene has remained unsettled. The 5'-region of the gene contains a SNP (rs3130453) that controls a 5'-extended open reading frame and thus the translation of alternative isoforms. We have now compared the function of two CCHCR1 isoforms: the novel longer isoform 1 and the previously studied isoform 3. In samples of Finnish and Swedish families, the allele generating only isoform 3 shows association with psoriasis (P<10(-7)). Both isoforms localize at the centrosome, a cell organelle playing a role in cell division. In stably transfected cells the isoform 3 affects cell proliferation and with the CCHCR1*WWCC allele, also apoptosis. Furthermore, cells overexpressing CCHCR1 show isoform- and haplotype-specific influences in the cell size and shape and alterations in the organization and expression of the cytoskeletal proteins actin, vimentin, and cytokeratins. The isoform 1 with the non-risk allele induces the expression of keratin 17, a hallmark for psoriasis; the silencing of CCHCR1 reduces its expression in HEK293 cells. CCHCR1 also regulates EGF-induced STAT3 activation in an isoform-specific manner: the tyrosine phosphorylation of STAT3 is disturbed in isoform 3-transfected cells. The centrosomal localization of CCHCR1 provides a connection to the abnormal cell proliferation and offers a link to possible cellular pathways altered in psoriasis.

CCHCR1 is up-regulated in skin cancer and associated with EGFR expression.

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30-80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.

Matrix metalloproteinase-21 expression is associated with keratinocyte differentiation and upregulated by retinoic acid in HaCaT cells.

In the skin, expression of several matrix metalloproteinases (MMPs) occurs in response to tissue injury, tumorigenesis, angiogenesis, apoptosis, and inflammation. The recently cloned MMP-21 has been implicated in skin development and various epithelial cancers. In this study, we found that it is also expressed by differentiated keratinocytes (KCs) in various benign skin disorders, in which it was not associated with KC apoptosis or proliferation, and in organotypic cultures. Furthermore, MMP-21 was induced in keratinocytes in association with increased calcium and presence of the differentiation marker filaggrin. In stably transfected A431 and HEK293 cell lines, MMP-21 increased invasion of cells but did not associate with increased apoptosis, proliferation, or epithelial-to-mesenchymal transition. Of various agents tested in HaCaT cell cultures, only retinoic acid (10(-6) M) and staurosporine (2.5 x 10(-8) M) upregulated MMP-21 mRNA and protein expression, whereas tumor promoters, hormones, or dexamethasone were without effect. Our results suggest that MMP-21 may be an important protease in the terminal differentiation of keratinocytes.