Spr1 Gene Research Articles

Functional analysis of Agaricus bisporus serine proteinase 1 reveals roles in utilization of humic rich substrates and adaptation to the leaf-litter ecological niche.

Summary Agaricus bisporus is a secondary decomposer fungus and an excellent model for the adaptation, persistence and growth of fungi in humic‐rich environments such as soils of temperate woodland and pastures. The A. bisporus serine proteinase SPR1 is induced by humic acids and is highly expressed during growth on compost. Three Spr1 gene silencing cassettes were constructed around sense, antisense and non‐translatable‐stop strategies (pGRsensehph, pGRantihph and pGRstophph). Transformation of A. bisporus with these cassettes generated cultures showing a reduction in extracellular proteinase activity as demonstrated by the reduction, or abolition, of a clearing zone on plate‐based bioassays. These lines were then assessed by detailed enzyme assay, RT‐qPCR and fruiting. Serine proteinase activity in liquid cultures was reduced in 83% of transformants. RT‐qPCR showed reduced Spr1 mRNA levels in all transformants analysed, and these correlated with reduced enzyme activity. When fruiting was induced, highly‐silenced transformant AS5 failed to colonize the compost, whilst for those that did colonize the compost, 60% gave a reduction in mushroom yield. Transcriptional, biochemical and developmental observations, demonstrate that SPR1 has an important role in nutrient acquisition in compost and that SPR1 is a key enzyme in the adaptation of Agaricus to the humic‐rich ecological niche formed during biomass degradation.

The Role of Fungi in Sustainable Agriculture and Energy

We have cloned and characterized spr1, a putative serine protease gene, from a nematode-trapping fungus, Monacrosporium megalosporum. The gene was present as a single copy in the genome. The predicted protein sequence of spr1 is homologous to the putative cuticle-degrading serine proteases PII and Azo1 from the nematode-trapping fungus, Arthrobotrys oligospora. In the 5' untranslated region near the initiation codon, consensus sequences to an AreA binding site, a well-known mediator of nitrogen metabolite repression in the fungus Aspergillus nidulans, a CreA binding site, a carbon response regulator in A. nidulans, and a PacC binding site, a transcription factor that responds to ambient pH signals in A. nidulans were found. However, spr1 was not regulated by carbon or nitrogen source, and exogenous protein did not induce expression of spr1. The transcription of the spr1 gene of this fungus was significantly affected by ambient pH. Based on RT-PCR, the product of the spr1 gene was not transcribed at pH 4, whereas under alkaline conditions such as pH 8 and 9, the spr1 gene was transcribed well. These results indicate that the spr1 gene is controlled only by a PacC homologue. Moreover, the expression profile of the spr1 gene corresponded with the pH-dependent physiology of this fungus.

Ambient pH signaling regulates expression of the serine protease gene ( spr1) in pine wilt nematode-trapping fungus, Monacrosporium megalosporum

We have cloned and characterized spr1, a putative serine protease gene, from a nematode-trapping fungus, Monacrosporium megalosporum. The gene was present as a single copy in the genome. The predicted protein sequence of spr1 is homologous to the putative cuticle-degrading serine proteases PII and Azo1 from the nematode-trapping fungus, Arthrobotrys oligospora. In the 5' untranslated region near the initiation codon, consensus sequences to an AreA binding site, a well-known mediator of nitrogen metabolite repression in the fungus Aspergillus nidulans, a CreA binding site, a carbon response regulator in A. nidulans, and a PacC binding site, a transcription factor that responds to ambient pH signals in A. nidulans were found. However, spr1 was not regulated by carbon or nitrogen source, and exogenous protein did not induce expression of spr1. The transcription of the spr1 gene of this fungus was significantly affected by ambient pH. Based on RT-PCR, the product of the spr1 gene was not transcribed at pH 4, whereas under alkaline conditions such as pH 8 and 9, the spr1 gene was transcribed well. These results indicate that the spr1 gene is controlled only by a PacC homologue. Moreover, the expression profile of the spr1 gene corresponded with the pH-dependent physiology of this fungus.

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion.

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.

SPR1 gene near HLA-C is unlikely to be a psoriasis susceptibility gene.

Although genetics analyses have identified the HLA-Cw6 allele to be the major risk allele for psoriasis vulgaris (PV) in many racial groups, it has been proposed that other putative genes near the HLA-C locus are involved in PV susceptibility and that the association of Cw6 is a result of linkage disequilibrium. The SPR1 gene, a predicted gene located 128 kb telomeric to the HLA-C locus, is considered to be one potential candidate gene of PV. Until now, no association study of the SPR1 gene has been conducted on psoriasis patients. We investigated the SPR1 gene for disease association by direct sequencing of the SPR1 gene in 116 Chinese patients with PV and 116 normal subjects. Genotyping for HLA-Cw6 was also carried out using polymerase chain reaction/restriction fragment length polymorphism. Significant increase of the HLA-Cw6 allele was found in psoriasis patients (32.8% vs. 13.8%, P = 0.001). We found that the SPR1 gene is a highly polymorphic gene containing 13 single nucleotide polymorphisms (SNPs), two of which have not been previously reported, and four SNPs cause amino acid change. No significantly different allelic distribution of 13 SPR1 SNPs could be found between the patients with PV and controls after correction for multiple testing. If the frequencies of SPR1 SNPs were compared between the early onset psoriatics and control subjects, early onset patients were more likely to have G allele at position 988 (60% vs. 35.3%, P = 0.001). However, the significance disappeared upon stratification for the Cw6 status. Haplotype-based association analysis showed two susceptibility haplotypes (types 8 and 19) in early onset psoriasis patients. Nonetheless, the significance also disappeared after stratification of the Cw6 status. Our results suggest that HLA-Cw6 remains the major risk allele in Chinese psoriatics, and that the SPR1 gene might not play an important role in the causation of PV.

Expression and regulation of a molecular marker, SPR1, in multistep bronchial carcinogenesis.

A small proline-rich protein, SPR1, is overexpressed in squamous metaplasia of bronchial epithelium. We studied the expression and regulation of SPR1 in a series of human bronchial epithelial cell lines representing a model of multistep bronchial carcinogenesis. These cell lines included a primary culture of tracheobronchial epithelial cells (HTBE), a papilloma virus-transformed tracheobronchial epithelial cell line (HBE1), a cell line selected from HBE1 by a tobacco carcinogen and a phorbol ester (HBE1-C), a simian virus-transformed bronchial epithelial cell line (BEAS-2B), and a lung carcinoma cell line (H460). Different tumorigenic potentials of these cell lines were indicated by graded levels of telomerase activity. Concomitant with squamous transformation, there was an increase in SPR1 expression in HTBE, HBE1, and HBE1-C that was reversible by vitamin A. With progression of tumorigenicity, there was a marked reduction in SPR1 expression in BEAS-2B and a total loss of expression in H460. In these latter cell lines representing advanced malignant transformation, there was a loss of up- and downregulation, respectively, by the phorbol ester and vitamin A. Transfection study with chimeric constructs of the SPR1 promoter and a reporter gene showed that the dysregulation of SPR1 expression in malignant transformation was a result of perturbation of the basal and enhancer elements of the first 162 nucleotides in the 5'-flanking promoter region of the SPR1 gene. These findings suggest an association of transcriptional dysregulation of the SPR1 gene with multistep bronchial carcinogenesis.

Re-expression of SPR1 in Breast Cancer Cells by Phorbol 12-Myristate 13-Acetate (PMA) or UV Irradiation Is Mediated by the AP-1 Binding Site in the SPR1 Promoter

Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation. The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. The mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the -619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream cis-acting promoter elements were identified. Our data indicate that the AP-1 element located between -139 and -133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 35-bp element located from -178 to -139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

Expression of the spr1 gene in cultured tracheal epithelial cells and its regulation by retinoids before and after confluence.

The absence of vitamin A or vitamin A derivatives in culture media promotes squamous cell differentiation of tracheobronchial epithelial cells. This is especially true for the expression of a small proline-rich protein (20K; 98 amino acids) in pig trachea epithelial cells. Multigene families encode different small proline-rich proteins in different species, and these proteins are possible markers for squamous cell differentiation. 20K mRNA and 20K protein were detected in cells within 4 and 5 days in culture, respectively, when cells reached about 50% confluence, and expression increase 12-fold during cell proliferation until cells reached 100% confluence. Arotinoid (10(-9)M), a synthetic retinoid, essentially totally inhibited expression of 20K mRNA in proliferating tracheobronchial cells within 3 days of treatment while 20K protein levels were only decreased 4-fold after 5 days. However, if cells were exposed to arotinoid 3 days after reaching confluent growth, the levels of either 20K mRNA or 20K protein were unchanged. Cells exposed to arotinoid from the onset of culturing, and then removal of the retinoid from proliferating cells resulted in the expression of 20K mRNA and protein after 4 and 5 days as observed previously. 20K mRNA was not detected in cells that had been continuously exposed to arotinoid from the start of culture until 3 days post confluence, even 10 days following removal of arotinoid. Our results strongly suggest that the growth phase and state of cell differentiation greatly affect the response of these epithelial cells to vitamin A derivatives.

Expression of Human Squamous Cell Differentiation Marker, SPR1, in Tracheobronchial Epithelium Depends on JUN and TRE Motifs

Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by co-transfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5'-flanking region contains the basal promoter activity, while the region between -162 and -96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: -141 TRE, -131 GT, -123 ETS-like, and -111 TRE-like motifs and in the enhanced protections in -141 TRE and -111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both -141 TRE and -111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the -111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.

Expression of a squamous cell marker, the spr1 gene, is posttranscriptionally down-regulated by retinol in airway epithelium.

Vitamin A (retinol) is required for the normal mucociliary differentiation of respiratory epithelium. A depletion of vitamin A promotes squamous cell metaplasia. To understand how vitamin A suppresses squamous cell differentiation, the expression of a squamous cell differentiation marker, the small proline-rich protein gene (spr1), was studied in cultured monkey tracheobronchial epithelial (TBE) cells. The expression of the spr1 gene was inhibited about 40 fold by retinol. The mRNA levels of the spr1 gene started to decline within 6 h of retinol treatment and reached a minimum level after 7 days. The inhibition by retinol was concentration dependent and did not require concurrent protein synthesis. The inhibition of the spr1 mRNA by retinol was not due to a decrease in the transcription rate of its gene but due to a decrease in its stability, as determined by nuclear run-on assays and mRNA half-life measurement, respectively. This result was further supported by a DNA transfection study using a chimeric construct containing the spr1 promoter region and the chloramphenicol acetyltransferase (CAT) reporter gene. The CAT activity in transfected cells was not inhibited by retinol. These results suggest that spr1 gene expression is posttranscriptionally down-regulated by retinol.

Gene Modulation Accompanying Differentiation of Normal versus Malignant Keratinocytes

Using tetradecanoylphorbol 13-acetate (TPA), a known inducer of epithelial cell differentiation, and Northern blot analysis, we investigated in normal versus well-differentiated malignant keratinocytes the modulation of genes implicated in their growth or differentiated function. In normal keratinocytes transient c-fos induction was detected within 30 min after stimulation and was followed by rapid down regulation of the proto-oncogenes c-myc and epidermal growth factor receptor. Within hours after stimulation mRNA levels for three well-characterized differentiated keratinocyte products, involucrin, interleukin-1β (IL1-β), and fibronectin, were induced, as were those for a growth arrest and DNA damage-inducible (GADD 153) gene and a small proline-rich (SPR1) gene, both known to be associated with differentiation but with of as yet unknown functions. Heat shock protein 70 gene was initially down regulated and was induced only after 48 h. The well-differentiated malignant keratinocyte cell line differed in that the c-fos, GADD, SPR1, and IL1-β genes had several-fold higher induction, but involucrin mRNA was undetectable and fibronectin mRNA was only minimally induced after TPA stimulation. Malignant cells reached terminal differentiation faster than normal keratinocytes as measured by inability to exclude trypan blue dye, and in situ hybridization using a riboprobe for the differentiation-associated SPR1 gene showed that normal keratinocytes constitutively express this transcript while malignant keratinocytes with virtually identical morphology and growth rate do not. These studies greatly expand our understanding of gene activation and down regulation during normal keratinocyte differentiation and imply that a malignant cell line, even when retaining the phenotype of normal cells, differs in its response to outside stimuli, furnishing at best an imperfect model for investigating the molecular mechanisms of cellular differentiation.

Isolation and characterization of the human spr1 gene and its regulation of expression by phorbol ester and cyclic AMP

The small proline-rich protein gene (spr1) is a marker whose expression is frequently associated with squamous cell differentiation. We observed that the expression of the spr1 gene is strongly induced by phorbol 12-myristate 13-acetate (PMA). Both the time course result and the nuclear run-on transcriptional assay suggested that the regulation of spr1 expression by PMA is controlled at the transcriptional level. To understand the nature of this regulation, human genomic clones of the spr1 gene were isolated. DNA sequence analysis revealed that the human spr1 gene contains two exons and a single intron located within the 5'-untranslated region. An AP-1 binding site (TGAGTCA) is found at -142, and a putative cyclic AMP-responsive element (TGAGGTCA) at -597 base pairs upstream of the transcription start site. A chimeric construct containing the 5'-flanking region of the spr1 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect HeLa cells or monkey primary TBE cells. The CAT activity in transfected cells is stimulated 7.5-11-fold by PMA, and the stimulation is inhibited by a protein kinase C inhibitor or by pretreating cells with PMA to down-regulate the protein kinase C activity. The CAT activity is also stimulated 3.5-fold by dibutyryl cyclic AMP, a protein kinase A activator. The stimulations by PMA and cAMP are additive. These results suggest that protein kinase C and probably protein kinase A play important roles in regulating the transcription of the spr1 gene.

An unusual expression of a squamous cell marker, small proline-rich protein gene, in tracheobronchial epithelium: differential regulation and gene mapping.

An unusual expression of a putative squamous cell marker, small proline-rich protein (spr1), in mucociliary epithelial cells of conducting airways was demonstrated in a serum-free culture system. A cDNA clone was isolated from the cDNA library of monkey tracheobronchial epithelial (TBE) cells by differential hybridization. This cDNA clone, MT5, exhibited 98% homology to a DNA sequence obtained from human keratinocytes treated with either UV light or phorbol esters (T. Kartasova et al., 1988, Mol. Cell. Biol. 8:2195-2230). The predicted peptide of MT5 is unusual for its high content of proline (29%), glutamine (18%), and cysteine (9%) and its repeated PKVPEPC units. The level of spr1 mRNA in cultured cells was inhibited more than 90% by vitamin A. In contrast, phorbol 12-myristate 13-acetate (PMA) stimulated the level of spr1 mRNA by 3- to 8-fold. This differential regulation coincided with the effects of these chemicals on the cornification of cultured TBE cells. Using MT5 as a probe, we have localized the tracheal spr1 gene on the human chromosome 1 by a Southern blot analysis using a panel of human-rodent somatic cell hybrid DNAs. The gene was further sublocalized to bands q22-23 by in situ hybridization.