Regulation of alternative splicing by PTB and associated factors.

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

Polypyrimidine tract binding protein functions as a repressor to regulate alternative splicing of alpha-actinin mutally exclusive exons.

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB.

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein.

PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.

Alternative promoter usage and mRNA splicing pathways for parathyroid hormone-related protein in normal tissues and tumours.

The parathyroid hormone-related protein (PTHrP) gene consists of nine exons and allows the production of multiple PTHrP mRNA species via the use of three promoters and 5' and 3' alternative splicing; as a result of 3' alternative splicing one of three protein isoforms may be produced. This organisation has potential for tissue-specific splicing patterns. We examined PTHrP mRNA expression and splicing patterns in a series of tumours and normal tissues, using the sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique. Use of promoter 3 and mRNA specifying the 141 amino acid PTHrP isoform were detected in all samples. Transcripts encoding the 139 amino acid isoform were detected in all but two samples. Use of promoters 1 and 2 was less widespread as was detection of mRNA encoding the 173 amino acid isoform. While different PTHrP splicing patterns were observed between tumours, no tissue- or tumour-specific transcripts were detected. In comparing normal and tumour tissue from the same patient, an increase in the number of promoters utilised was observed in the tumour tissue. Furthermore, mRNA for the PTH/PTHrP receptor was detected in all samples, thus the PTHrP produced by these tumours may potentially act in an autocrine or paracrine fashion.

Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability.

Epidermal growth factor (EGF) produced rapid and striking effects on parathyroid hormone-related protein (PTHrP) gene expression in the immortalized human keratinocyte cell line, HaCaT. Steady-state levels of PTHrP mRNA and secreted PTHrP were increased 10-fold by maximally effective concentrations of EGF. EGF increased both PTHrP gene transcription and PTHrP mRNA stability. Nuclear run-on assays demonstrated a 4-fold increase in transcriptional rate in EGF-stimulated cells while transient transfection analysis indicated that the action of EGF on transcription involved both the GC-rich promoter, P2, and the downstream TATA promoter, P3, but apparently not the upstream TATA promoter, P1. In experiments where EGF treatment produced more stable PTHrP transcripts, the half-life of c-fos mRNA was unaltered, suggesting a relatively specific effect of EGF. Moreover, only those species of PTHrP mRNA containing two of the alternative 3' exons (exons VII and VIII) were stable, those containing exon IX were not. Reverse-transcription PCR demonstrated that EGF produced differential increases in the abundance of PTHrP mRNA species initiated by the three PTHrP promoters. The major effect was seen on the abundance of transcripts initiated by P1 and P2, with less marked regulation of P3-initiated transcripts. Thus EGF regulation of PTHrP gene expression in HaCaT cells is multifactorial and the combination of its actions at the 5' and 3' ends of the gene favours the accumulation of subpopulations of PTHrP mRNA containing exons I, VII and VIII.

Dexamethasone regulation of parathyroid hormone-related protein (PTHrP) expression in a squamous cancer cell line.

Dexamethasone regulation of PTHrP expression has been studied in an epidermal squamous cancer cell line COLO 16, which secretes immunoreactive PTHrP into conditioned medium. Dexamethasone was found to suppress PTHrP expression in a time- and dose-dependent manner, which was reversible upon removal of dexamethasone. The half-maximal effective concentration of dexamethasone was 1 nM and an effect of dexamethasone on PTHrP mRNA was first observed after 2 h of treatment, with maximal inhibition by 6 h. Dexamethasone action on PTHrP expression was steroid specific since progestin, 5alpha-dihydroxytestosterone and oestrogen did not regulate PTHrP expression in COLO 16 cells. The gluocorticoid/progesterone receptor antagonist RU486 inhibited the dexamethasone effect, indicating glucocorticoid receptor-mediated regulation of PTHrP expression. The half-life of PTHrP mRNA in COLO 16 cells was approximately 120 min and was not altered by treatment of cells with dexamethasone. Nuclear run-on assays revealed that dexamethasone reduced PTHrP gene transcription in COLO 16 cells. Transient transfection assays with a series of reporter gene constructs encompassing 3.5 kb of the 5' end of the PTHrP gene failed to identify a region of the gene responsible for glucocorticoid down-regulation. PCR of reverse-transcribed RNA from COLO 16 cells revealed that dexamethasone down-regulated transcripts driven from all three promoters (i.e., the TATA promoters 5' to exons I and IV and the GC-rich promoter 5' to exon III) of the human PTHrP gene.

Localization of parathyroid hormone-related protein mRNA expression in breast cancer and metastatic lesions by in situ hybridization.

Parathyroid hormone-related protein (PTHrP) has been identified immunohistochemically in 60% of breast carcinoma and in 92% of breast cancer metastases in bone. To establish whether the localization of the PTHrP antigen reflects protein synthesis and also to investigate the role of PTHrP in metastatic disease, as part of an ongoing study, we used in situ hybridization to study the localization of PTHrP mRNA in a retrospective series of primary breast tumors and their metastatic lesions. Paraffin sections of 17 primary and 26 metastatic lesions, 11 of which were in bone, were available for the study: 10 of the 17 (59%) primary lesions, 8 of 11 (73%) breast cancer metastases to bone, and 3 of 15 (20%) metastases to non-bone sites showed specific localization of PTHrP mRNA. These findings establish that PTHrP is commonly synthesized by primary breast cancers and support previous immunohistochemical studies reporting a higher incidence of PTHrP-positive tumor cells in skeletal metastases than in nonskeletal metastases.

Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia.

PTH-related protein (PTHrP) is commonly produced by squamous cell carcinomata and is the mediator of the PTH-like features of humoral hypercalcemia of malignancy. It has also been implicated in calcium regulation during fetal development. In this study immunohistochemical techniques, using rabbit polyclonal antibodies to synthetic PTHrP peptides, have been used to localize PTHrP in human fetal tissues from one fetus of 7 weeks and two of approximately 18 and 20 weeks gestation, respectively, in order to identify sites of potential functional significance. PTHrP immunoreactivity was identified in epithelia from many sources, including skin, bronchus, pancreas, pharynx, gut, stomach, and renal pelvis. Thyroid and parathyroid glands, which develop from epithelial origins, also stained positive for PTHrP, as did kidney collecting tubules, adrenal tissue, and skeletal and smooth muscle. PTHrP immunoreactivity was also located in developing long bones and calvaria, where it may have relevance in bone turnover during fetal development. The role of PTHrP at these locations remains to be elucidated, but the identification of specific PTHrP immunoreactivity in fetal epithelia is consistent with PTHrP production by cancers of epithelial origin and supports the hypothesis that PTHrP may have a role in epithelial growth and differentiation.

Ontogeny of parathyroid hormone-related protein in the ovine parathyroid gland.

PTH-related protein (PTHrP) has been implicated in calcium regulation during fetal life. In this study the ontogeny of PTHrP was examined in ovine parathyroid glands. Immunohistochemical techniques, Western blot analysis, and a RIA with antisera raised against synthetic fragments of human (h) PTHrP (i.e. 1-34, 1-40, 50-69, and 107-141) were used to detect the presence of immunoreactive PTHrP in parathyroid glands from fetal and neonatal lambs and maternal ewes. Positive immunostaining for PTHrP was observed in fetal (from 116 days of gestation) and lamb (up to 180 days post birth) but not maternal parathyroid glands with the PTHrP(50-69) antiserum. Fetal and lamb parathyroid glands consisted entirely of one cell type in which PTHrP immunoreactivity to PTHrP(50-69) antiserum was found. In contrast, immunoreactivity to PTHrP could not be detected in sections of fetal, lamb, or maternal parathyroid glands with antisera raised against PTHrP(1-34) or PTHrP(107-141). However, PTHrP immunoreactivity in urea/acid extracts of newborn lamb parathyroid glands could be detected by Western blot analysis and RIA with antisera raised against the N-terminal portion of PTHrP. Western blot analysis with the PTHrP(1-34) antisera revealed that urea/acid extracts of newborn lamb parathyroid glands contained a substance with a mol wt of 14.4K, which corresponded in size to that of hPTHrP(1-84). Newborn lamb parathyroid glands contained 0.35 ng PTHrP/micrograms extract, whereas maternal parathyroid glands contained only 0.035 ng PTHrP/micrograms extract when tested in a RIA employing recombinant hPTHrP(1-84) as standard and an antibody raised against hPTHrP(1-40). The detection of immunoreactive PTHrP in the developing ovine parathyroid gland provides further evidence to support the suggestion that PTHrP produced in the parathyroid gland is involved in the normal hormonal regulation of calcium metabolism in the mammalian fetus and neonate.

Localization of parathyroid hormone-related protein in breast cancer metastases: increased incidence in bone compared with other sites.

Parathyroid hormone-related protein (PTHrP) has recently been identified in 60% of a series of primary breast cancers. The detection of a bone-resorbing factor in tumors with a propensity to metastasize to bone prompted study of PTHrP in breast cancer metastasis. PTHrP was localized by immunohistology in 12 of 13 (92%) breast cancer metastases in bone and in 3 of 18 (17%) metastases in non-bone sites. The statistical difference was highly significant (P less than 0.0001). Production of PTHrP as a bone-resorbing agent may contribute to the ability of breast cancers to grow as bone metastases.

Immunohistochemical localization of parathyroid hormone-related protein in human breast cancer.

Parathyroid hormone-related protein (PTHrP) is known to be a causative factor in humoral hypercalcemia of malignancy. A polyclonal rabbit antiserum directed against the amino-terminal region of the protein and immunoperoxidase methods have been used to locate the presence of PTHrP in a series of 102 consecutive invasive breast tumors removed surgically from normocalcemic women. Positive PTHrP staining was detected in 60% of the tumors but not in the accompanying normal breast tissue. Positive staining was related to the progesterone receptor status of the tumor (P = 0.039) and to the prognostic index of the patient (P = 0.046) and not to estrogen receptor status, patient age, tumor size, histological grade, or nodal status.

Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia.

Parathyroid hormone-related protein (PTHrP) is invoked as the cause of humoral hypercalcaemia of malignancy (HHM); it is contained in the keratinocyte layer of normal skin; and there is evidence that is is produced by fetal parathyroids. Antibodies against synthetic PTHrP peptides have been raised in rabbits and sheep. This immunohistochemical study has found that primary parathyroid adenomata and hyperplastic glands from patients with chronic renal failure stain positively with antisera against PTHrP(1-34) and PTHrP(50-69). Primary hyperplastic glands are negative. No staining with anti-PTHrP(106-141) antiserum could be detected immunohistochemically in any of the parathyroid adenomata or hyperplasia.