Full-length Fragments Research Articles

Expression of diphtheria toxin inStreptococcus mutansand induction of toxin-neutralizing antisera

The nontoxic full-length diphtheria toxin (DTX), fragment A (DTA), and fragment B (DTB) were each genetically fused to the major surface protein antigen P1 (SpaP) of Streptococcus mutans. Repeated attempts to express the recombinant DTX and DTB in the live oral vaccine candidate Streptococcus gordonii were unsuccessful, whereas DTA could be readily expressed in this bacterium. However, the recombinant DTX, DTB, and DTA could be expressed in the related oral bacterium S. mutans. Western blotting and enzyme-linked immunosorbant assay (ELISA) using anti-DTX and anti-P1 antibodies demonstrated the expression of the three fusion proteins in S. mutans. Mouse antisera raised against the recombinant S. mutans recognized the native DTX in Western immunoblotting. The antibodies raised against S. mutans expressing the recombinant DTX and DTA neutralized the cytotoxicity of the native toxin in a Vero cell assay, but the neutralization titers were relatively low. The potential of using S. gordonii as a live vaccine against diphtheria faces major challenges in the expression of DTX in this organism and in the induction of high-titer toxin-neutralizing antibodies.

An automated genotyping system for analysis of HIV-1 and other microbial sequences

Genetic analysis of HIV-1 is important not only for vaccine development, but also to guide treatment strategies, track the emergence of new viral variants and ensure that diagnostic assays are contemporary and fully optimized. However, most genotyping methods are laborious and complex, and involve the use of multiple software applications. Here, we describe the development of an automated genotyping system that can be easily applied to HIV-1 and other rapidly evolving viral pathogens. The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with boot-scanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5-100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database.

A versatile promoter for the expression of proteins in glandular and non-glandular trichomes from a variety of plants

A DNA regulatory fragment was isolated from the promoter region of the OASA1 gene, encoding the cytosolic O-acetylserine(thiol)lyase enzyme that is highly expressed in Arabidopsis thaliana trichomes. This DNA fragment has been named an ATP fragment and comprises 1435 bp of the genomic region upstream of the OASA1 gene and 375 bp of the transcriptional initiation start site containing the first intron of the gene. The ATP fragment, fused to the green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes, is able to drive high-level gene expression in A. thaliana trichomes. Deletion analysis of the ATP fragment determined that the region from -266 to -66 contains regulatory elements required for trichome expression. In addition, the region from +112 to +375, comprising the first intronic region of the gene, is also essential for trichome gene expression. Expression of the full-length ATP fragment in tobacco and peppermint shows that this fragment is also able to drive expression in glandular trichomes and suggests additional biotechnological applications for this promoter.

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5′-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.

Bone Morphogenic Protein-7 Induces Mesenchymal to Epithelial Transition in Adult Renal Fibroblasts and Facilitates Regeneration of Injured Kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.

Hydrolysis of the amyloid β‐peptide (Aβ) 1–40 between Asp23–Val24 produces non‐aggregating fragments. An electrospray mass spectrometric study

The aggregation of full-length (residues 1-40) amyloid beta-peptide (A beta) and fragments corresponding to residues 1-23 and 24-40 was studied by electrospray mass spectrometry, using gramicidin as a non-aggregating reference. Following a lag period, A beta(1-40) at 140 microM concentration aggregates with apparent first-order kinetics. Under acidic conditions A beta(1-40) undergoes spontaneous cleavage between Asp23-Val24 and to a lesser extent also at two other Asp-X motifs. Incubation in acidic H(2)18O showed incorporation of 18O in fragment A beta(1-23), confirming that the Asp23-Val24 peptide bond had been hydrolyzed. Incubation of synthetic A beta(1-23) and A beta(24-40) peptides with A beta(1-40) showed that A beta(24-40) remained in solution for several months, that A beta(1-23) partly disappeared from solution, whereas A beta(1-40) completely disappeared. Further, treatment of sedimentable aggregates formed after co-incubation of the three peptides with hexafluoro-2-propanol or formic acid recovered the intensity of A beta(1-40). These data support previous studies showing that the region of A beta encompassing residues 16-24 is necessary for aggregation into amyloid fibrils.

The Tandem BRCT Domains of Ect2 Are Required for Both Negative and Positive Regulation of Ect2 in Cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.

Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

We have previously shown that avian reovirus (ARV) σA and σNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on σA (I and II) and three epitopes (A, B, and C) on σNS. To further define the location of epitopes on σA and σNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of σA and σNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on σA was located at amino acid residues 340QWVMAGLVSAA 350 and epitope B on σNS at amino acid residues 180MLDMVDGRP 188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of σA and σNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete σNS but not with truncated σNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on σNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on σA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the σNS with MAb 1F9. The σNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on σNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of σNS.

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.

The Crystal Structure of RhoA in Complex with the DH/PH Fragment of PDZRhoGEF, an Activator of the Ca2+ Sensitization Pathway in Smooth Muscle

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.

PTH revisited

Recent investigations of parathyroid hormone (PTH) have advanced our understanding of its circulating forms as well as its action. It is now clear that first-generation immunoradiometric assays of so-called intact "PTH" not only measured full-length PTH(1-84) but also recognized large PTH fragments lacking the amino-terminus. New, second generation assays detect only full-length PTH. Under diverse pathological settings, second generation assays display lower levels of PTH (1-84). By measuring full-length PTH (bioactive PTH) and the combined full-length plus amino-terminal PTH fragments, the amount of non-PTH(1-84) in circulation can be estimated. The primary amino-terminal fragment is likely to be PTH(7-84). A considerable controversy surrounds the pathological significance of PTH(7-84) and its relation to adynamic bone disease. While these findings were emerging, other work uncovered the apparent basis by which PTH receptors signal through cAMP in some instances but through Ca/inositol phosphate in others. This signaling switch is dictated by the cytoplasmic adapter protein NHERF1 (EBP50), which is expressed in a cell-selective fashion. Other provocative findings may provide a means of unifying determinations of PTH(7-84) with the effects of NHERF1 on PTH receptor signaling. These latter studies reveal that in cells expressing NHERF1, PTH(7-84) has no effect on PTH receptor signaling or internalization. However, in cells lacking or expressing low levels of NHERF1, PTH(7-84) internalizes the PTH receptor without accompanying activation. Together, these findings suggest that the accumulation of PTH(7-84) in renal failure may lead to PTH resistance by internalizing and down-regulating PTH receptors.

Intrinsic versus mutation dependent instability/flexibility: a comparative analysis of the structure and dynamics of wild-type transthyretin and its pathogenic variants

Transthyretin (TTR) is one of the about 20 known human proteins associated with amyloidosis which is characterized by the accumulation of amyloid fibrils in tissues or extracellular matrix surrounding vital organs. Unlike Alzheimer’s fibrils that comprise a fragment of a large precursor protein, TTR amyloid fibrils are composed of both full-length protein and fragments of the molecule. The native state of TTR is a homotetramer with eight β-strands organized into a β-sandwich in each monomer. To elucidate the structural reorganization mechanisms preceding amyloid formation, it is important to characterize the dynamic features of the wild-type native state as well as to reveal the influence of disease-associated mutations on the structure and dynamics. Molecular dynamics (MD) simulations complement X-ray crystallography and D–H exchange to capture the intrinsically unstable/flexible sites of the wild-type as well as the mutation dependent unstable sites of the pathogenic variants. Our results of MD simulations have shown that the Leu55 → Pro (L55P) mutation occurs in an intrinsically unstable site, leading to substantial local and global structural changes. This observation supports the early speculation that the C-strand-loop–D-strand rearrangement leads to the formation of amyloidogenic intermediates. In addition to the D strand, the α-helical region and the strands at the monomer–monomer interface are also intrinsically unstable. The central channel of L55P-TTR undergoes opening and closing fluctuations, which may provide an explanation for the fact that while the mutation is far from the channel, the mutant shows a substantial low binding affinity of thyroxine.

Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3.

To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3.1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3.1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5% and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3.1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.

CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk

Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 receptor (N3 ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3 ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3 ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that are located outside the putative ligand binding domain (LBD) of the receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC, whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling activity. Ligand binding assays with soluble Delta1 and Jagged1 revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. All mutant receptors including Notch3 C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa receptor and S1-cleaved receptor fragments. Taken together, these data indicate that CADASIL-associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk, whereas they have similar effects on receptor maturation. Moreover, the data suggest that ligand-induced receptor shedding may not be required for N3 ECD deposition in CADASIL.

Interaction of filamin A with the integrin β7 cytoplasmic domain: role of alternative splicing and phosphorylation

Integrin–filamin binding plays an important role in adhesion-mediated control of the actin cytoskeleton. Here, using the interaction between recombinant fragments from the C-terminus of filamin A and the cytoplasmic tail of integrin β7 as a model, we report a negative regulatory role for filamin alternative splicing. Splice variant forms of filamin A lacking a 41-amino acid segment interacted more strongly than full-length fragments. In addition, we provide evidence that phosphorylation of the splice variant region is unlikely to represent the mechanism by which binding is reduced.

Regulation of ceruloplasmin gene in mammals.

A site of rat DNA (about 1800 b. p.) adjacent to the first ceruloplasmin gene contains, apart from regulatory sequences common for all eukaryotic promotors, cis-elements, which are potential binding sites for soluble nuclear receptors of some hormones. Sequences characteristic of genes expressed in liver cells and mammary gland cells during lactation were detected. Full-length fragment of this locus of ceruloplasmin gene (1800 b. p.) was synthesized by PCR and used in gel shift experiments. It was found that soluble proteins extracted from purified nuclei of mammary gland cells during lactation and from the liver of adult and newborn rats, contain proteins specifically interacting with the PCR product. A fragment of chromosome gene containing exons encoding the central part of rat ceruloplasmin was cloned in pTZ19 bacterial vector. Gel shift assay showed that the cloned fragment contained binding sites for specific transcription factor YY1, whose level in nuclear protein fractions varied during ontogeny (according to immunoblotting data). Monoclonal antibodies detected protein YY1 in the complex of cloned DNA-nuclear proteins. Possible mechanisms of tissue-specific regulation of ceruloplasmin gene varying during ontogeny are discussed.

First report of cassava mosaic disease and cassava mosaic geminiviruses in Gabon

Cassava mosaic disease (CMD), arguably Africa's greatest plant protection problem, has been known to occur in central/West Africa for more than 70 years. There is, however, no published record of the occurrence of CMD or the cassava mosaic geminiviruses (CMGs) that cause it from the central African country of Gabon. Significantly, however, the severe Uganda variant of East African cassava mosaic virus (EACMV-UG), associated with the African CMD pandemic, has been recorded from the Republic of Congo (Neuenschwander et al., 2002), bordering Gabon to the west. Cassava fields were examined at 55 sites throughout Gabon in April and July 2003, in order to identify the CMGs associated with CMD and to determine if the pandemic-associated EACMV-UG was present. At each site, 30 plants were examined and assessments made of CMD incidence, symptom severity and the abundance of the whitefly vector Bemisia tabaci. At least two virus-diseased leaf samples were collected from each field for subsequent virus diagnosis. DNA was extracted from these samples on the day of collection using the method of Dellaporta et al. (1983). Virus diagnoses were subsequently made from DNA samples using both specific primer PCR (Zhou et al., 1997) and RFLP analysis involving restriction digestion by EcoRV and MluI of near full-length DNA-A fragments amplified using abutting primer PCR with universal geminivirus primers (Briddon & Markham, 1994). ACMV was the most widely distributed CMG species, occurring at 53 of the 55 sites. EACMV was identified from a single site just south of Tchibanga in southern Gabon. EACMV-UG was detected in samples collected from 12 sites, all of which were in the eastern provinces of Haute-Ogooué and Ogooué-Ivindo. Of the 17 samples infected with EACMV-UG, 16 were mixed infections with ACMV. Symptom severity of plants infected by EACMV-UG (4·1) was significantly greater (χ2 = 44·4, d.f. = 3, P < 0·001) than that of plants infected by ACMV alone (2·8). Additionally, four of the five virus-sampled plants having current season whitefly-borne CMD were dual ACMV + EACMV-UG infections. These data comprise the first published record of CMGs in Gabon. The results also describe an early stage of spread of the EACMV-UG-associated CMD pandemic into eastern Gabon, which now represents the pandemic's westernmost ‘front’. Grant assistance from the Office for US Foreign Disaster Assistance and the technical help of Dr M. Tindo and Mr R. Obonyo are acknowledged.

Characterization of Four Autonomous Repression Domains in the Corepressor Receptor Interacting Protein 140

Receptor interacting protein (RIP) 140 is a corepressor that can be recruited to nuclear receptors by means of LXXLL motifs. We have characterized four distinct autonomous repression domains in RIP140, termed RD1-4, that are highly conserved in mammals and birds. RD1 at the N terminus represses transcription in the presence of trichostatin A, suggesting that it functions by a histone deacetylase (HDAC)-independent mechanism. The repressive activity of RD2 is dependent upon carboxyl-terminal binding protein recruitment to two specific binding sites. Use of specific inhibitors indicates that RD2, RD3, and RD4 are capable of functioning by HDAC-dependent and HDAC-independent mechanisms, depending upon cell type.

Requirement for proximal putative Sp1 and AP-2 cis-deoxyribonucleic acid elements in mediating basal and luteinizing hormone- and insulin-dependent in vitro transcriptional activation of the CYP17 gene in porcine theca cells.

The cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17) enzyme catalyzes the first committed step in androgen biosynthesis. In primary cultures of immature swine theca cells, LH and insulin induce CYP17 mRNA and incompletely processed heteronuclear RNA supraadditively over 2-6 h. To monitor in vitro transcriptional control by these two physiological signals, we cloned a -976 to +31-bp 5'-upstream region of the homologous CYP17 gene and fused it to a cytoplasmically targeted firefly luciferase minigene (CYP17/luc). LH and insulin individually stimulated transcriptional activity of transiently transfected CYP17/luc in theca cells by 2.7 +/- 0.31- and 2.5 +/- 0.24-fold over basal, respectively, at an optimal concentration (both 100 ng/ml) and time (6 h; both P < 0.01). Combined peptidyl agonists stimulated CYP17/luc by 6.6 +/- 1.2-fold (P < 0.001). To identify possible LH- and insulin-sensitive cis-acting DNA regulatory regions, we prepared four deletional constructs, -839, -473, -174, and -75/+35 bp 5' upstream of the transcriptional start site. Deletion from -976 to -839 bp decreased basal transcriptional activity by 89% and that stimulated by LH, insulin, and both effectors by 82%, 91%, and 78%, respectively (each P < 0.01). Further deletion to -473 bp conferred partial responsiveness to combined hormone stimulation, suggesting an intervening inhibitory sequence. Truncation to -174 bp and more proximally reduced basal CYP17/luc activity and hormonal action by more than 95% (P < 0.001). The marked loss of combined LH and insulin stimulation caused by deleting the region between -473 and -175 bp suggested the possible relevance of partially overlapping specificity protein-1 (Sp1) and activating protein-2 (AP-2)-like binding sites located between -193 and -180 bp. Point mutation of the proximal Sp1-like element in full-length -976/+31 CYP17/luc impaired basal transcription minimally (by 21%; P = NS) and stimulation by LH (76%), insulin (67%), and combined peptides (54%) significantly (each P < 0.05 vs. wild type). Mutation of the AP-2 site alone decreased basal CYP17/luc activity nonsignificantly (by 25%), but repressed stimulated transcriptional responses prominently, viz. to LH (57%), insulin (77%), and both effectors (82%; each P < 0.025 vs. wild type). Mutation of both sites inhibited basal and hormonally stimulated CYP17/luc activity by more than 95% (P < 0.001). At the level of second messenger signaling, insulin did not potentiate LH-enhanced cAMP accumulation, whereas a stable cAMP analog mimicked LH action and augmented insulin's stimulation of full-length and deletional fragments of CYP17/luc. In summary, LH and insulin stimulate transcriptional activity of a -976/+31 bp 5'-upstream cis-acting region of the (porcine) CYP17 gene individually and jointly in primary cultures of theca cells. Maximal transcriptional responsiveness to these peptide hormones requires proximal Sp1 and AP-2-like sequences -193 to -180 bp 5' upstream of the transcriptional start site. Exogenous cAMP mimics transcriptional up-regulation by LH and interacts analogously with insulin. These data are consistent with convergent drive of CYP17 gene expression by cAMP-protein kinase A and insulinsignaling pathways in untransformed theca cells.

Insulin drives transcriptional activity of the CYP17 gene in primary cultures of swine theca cells.

Insulin stimulates androgen biosynthesis and the accumulation of CYP17 mRNA and heterogeneous nuclear (hn) RNA in primary cultures of immature swine theca cells. To further assess insulinomimetic transcriptional control, we subcloned 1.007 kilobases (kb) of the 5'-upstream region of the CYP17 gene (-976 to +31 base pairs [bp] to the transcriptional start site) into a firefly-luciferase reporter construct. Insulin drove transcriptional activity of this probe in a time- and dose-dependent fashion, with maximal stimulation of 2.7- to 3.2-fold after insulin exposure (100 ng/ml) for 6 h. Progressive deletional constructs -839, -473, -174, and -75/+31 bp delineated expected reduction in responsiveness, except paradoxical gain of basal CYP17 promoter activity by the -473/+31-bp sequence. The latter suggests a possible intervening inhibitory sequence. Elimination of all sequences 5'-upstream to -174 bp markedly reduced basal transcriptional activity and abolished insulin action. Point mutation of a presumptive Sp1-like element located within -193/-180 bp inhibited basal and insulin-stimulated luciferase activity of the full-length promoter fragment by 40% and 67%, respectively. Disruption of a contiguous presumptive AP-2 site produced a comparable outcome. Combined mutation of the Sp1 and AP-2-like elements eliminated basal and insulin-potentiated CYP17 promoter activity. By Western analysis, insulin augmented cognate receptor phosphoprotein concentrations by 31-fold within 10 min. Chemical inhibitors of MEK-activated ERK1/2 attenuated insulin-enhanced CYP17 transcriptional activity by 76-80%. In summary, insulin drives transcriptional activity of a 5'-upstream regulatory sequence (-976 to +31 bp) of the swine CYP17 gene in primary cultures of theca cells, under a minimal requirement for combined activity of proximal (-193/180 bp) Sp1 and AP-2-like elements.