T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and homeostasis of the sebaceous gland

The important role of epidermal appendages especially the sebaceous gland has only recently been recognized. In particular, it has been convincingly shown that normal development and maintenance of the sebaceous gland are required for skin homeostasis since atrophic sebaceous glands and disturbances in sebaceous lipid composition result in major defects of the physiological barrier and maintenance of the skin. Consequently, it is important to unravel the signaling network controlling proper sebaceous lineage differentiation in mammalian skin and to understand the underlying mechanisms leading to severe skin diseases, including abnormal proliferation and differentiation of the gland, defects of the lipid metabolism and barrier, as well as sebaceous tumor formation. Over the last years, results from transgenic and knock out mouse models manipulating distinct signaling pathways in the skin as well as the detailed analysis of human sebaceous gland-derived cell lines provided new insights into crucial mediators balancing proliferation and differentiation of the sebaceous gland. Here, we discuss our current knowledge of in vivo mechanisms of sebaceous gland development, maintenance and disorders and highlight recent contributions to the field of sebaceous gland biology.     

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.     

低分子量聚乙亚胺介导的基因转染系统及动物皮肤组织基因转染试验

将带有绿色荧光蛋白(GFP)报告基因的真核表达质粒与阳离子聚合物聚乙亚胺(PEI)结合,用肝癌细胞株CM7221实验,研究其转染效率及可能引起的细胞毒性;进一步用此PEI/DNA复合物转染小鼠皮肤组织,通过报告基因检测,研究转染基因的表达位置及持续表达时间。结果发现,低分子量PEI介导的细胞转染效率最高可达55%,转染效率与PEI结构无关,但是随着分子量的增加,转染活性略有下降。同时,随着分子量的增加,PEI对细胞的毒性也相应的加大;动物皮肤转染实验显示,转染24h后,GFP基因在皮肤组织的毛囊、汗腺、皮脂腺等处高效表达,表达可持续7天。表明低分子量PEI是低毒性、高转染效率的有用非病毒转染载体,能够在动物皮肤组织中进行基因转移,这对皮肤疾病的基因治疗具有潜在的应用价值。     

低分子量聚乙亚胺介导的基因转染系统及动物皮肤组织基因转染试验

将带有绿色荧光蛋白(GFP)报告基因的真核表达质粒与阳离子聚合物聚乙亚胺(PEI)结合,用肝癌细胞株CM7221实验,研究其转染效率及可能引起的细胞毒性;进一步用此PEI／DNA复合物转染小鼠皮肤组织,通过报告基因检测,研究转染基因的表达位置及持续表达时间。结果发现,低分子量PEI介导的细胞转染效率最高可达550%,转染效率与PEI结构无关,但是随着分子量的增加,转染活性略有下降。同时,随着分子量的增加,PEI对细胞的毒性也相应的加大;动物皮肤转染实验显示,转染24h后,GFP基因在皮肤组织的毛囊、汗腺、皮脂腺等处高效表达,表达可持续7天。表明低分子量PEI是低毒性、高转染效率的有用非病毒转染载体,能够在动物皮肤组织中进行基因转移,这对皮肤疾病的基因治疗具有潜在的应用价值。     

Molecular and biochemical characterization of a serine proteinase predominantly expressed in the medulla oblongata and cerebellar white matter of mouse brain

A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4-methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain.     

Characterization of mouse glandular kallikrein 24 expressed in testicular Leydig cells

Mouse kallikrein 24 is thought to encode a functional serine protease belonging to the mouse glandular kallikrein gene family. Preliminary results suggest that this kallikrein may play a role in testis function in adult mice. In order to obtain insights into its physiological functions, we undertook molecular and biochemical analyses of this enzyme. We cloned a cDNA for kallikrein 24 from the adult mouse testis cDNA library. Kallikrein 24 was expressed in the kidney, submandibular glands, ovary, epididymis, and testis of the mouse. In the testis, kallikrein 24 mRNA was detectable at 4 weeks of postnatal development, and became more prominent thereafter. The kallikrein 24 gene was expressed exclusively in the Leydig cells of adult mice. When Leydig cells isolated from a 2-week-old mouse testis were cultured in the presence of testosterone, kallikrein 24 expression was induced. Active recombinant enzyme showed trypsin-like specificity, favorably cleaving Arg-X bonds of synthetic peptide substrates. The enzymatic activity was strongly inhibited by typical serine protease inhibitors. Mouse kallikrein 24 degraded casein, gelatin, fibronectin and laminin. These results suggest that the enzyme may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis. The present findings should contribute to future physiological studies of this mouse testis protease.     

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.     

Subfamily of submaxillary gland-specific Mup genes: chromosomal linkage and sequence comparison with liver-specific Mup genes

Mouse major urinary proteins (MUPs) are encoded by a family of ca. 35 genes that are expressed in a tissue-specific manner in several secretory organs; in the liver, in the submaxillary, sublingual, parotid and lachrymal glands, and in the skin sebaceous glands. In this paper we describe the isolation of a Mup gene, Mup-1.5a, which is expressed predominantly in the submaxillary gland of BALB/c mice. We show that Mup-1.5a is a member of a subfamily consisting of two closely related genes, both of which are closely linked to the Mup-1 locus on mouse chromosome 4. Mup-1 is the locus of a class of Mup genes (Group 1) expressed in the liver. The complete nucleotide sequence of Mup-1.5a has been determined, and was compared to a previously sequenced Group 1 Mup gene. The comparison shows that the differentially expressed Mup genes are uniformly divergent in exons, introns and in their flanking sequences. The regions of homology extend at least 5 kb into the 5' flanking region of Mup genes.     

Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases

A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for beta-catenin and Notch signalling

Retinoic acid (RA) signalling is essential for epidermal differentiation; however, the mechanisms by which it acts are largely unexplored. Partitioning of RA between different nuclear receptors is regulated by RA-binding proteins. We show that cellular RA-binding proteins CRABP1 and CRABP2 and the fatty acid-binding protein FABP5 are dynamically expressed during skin development and in adult tissue. CRABP1 is expressed in embryonic dermis and in the stroma of skin tumours, but confined to the hair follicle dermal papilla in normal postnatal skin. CRABP2 and FABP5 are expressed in the differentiating cells of sebaceous gland, interfollicular epidermis and hair follicles, with FABP5 being a prominent marker of sebaceous glands and anagen follicle bulbs. All three proteins are upregulated in response to RA treatment or Notch activation and are negatively regulated by Wnt/β-catenin signalling. Ectopic follicles induced by β-catenin arise from areas of the sebaceous gland that have lost CRABP2 and FABP5; conversely, inhibition of hair follicle formation by N-terminally truncated Lef1 results in upregulation of CRABP2 and FABP5. Our findings demonstrate that there is dynamic regulation of RA signalling in different regions of the skin and provide evidence for interactions between the RA, β-catenin and Notch pathways.