Human Parathyroid Hormone-related Protein Gene Research Articles

1,25-Dihydroxyvitamin D 3 regulates PTHrP expression via transcriptional, post-transcriptional and post-translational pathways

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression. PTHrP expression is suppressed by 1,25-dihydroxyvitamin D 3 (1,25D). The aim of this study was to identify the pathways via which 1,25D exerts these effects. Our main findings are that 1,25D regulates PTHrP levels via multiple pathways in PC-3 and C4-2 (human prostate cancer) cell lines, and regulation is dependent on VDR expression. The human PTHrP gene has three promoters (P); PC-3 cells preferentially utilize P2 and P3, while C4-2 cells preferentially utilize P1. 1,25D regulates PTHrP transcriptional activity from both P1 and P3. The 1,25D-mediated decrease in PTHrP mRNA levels also involves a post-transcriptional pathway since 1,25D decreases PTHrP mRNA stability. 1,25D also suppresses PTHrP expression directly at the protein level by increasing its degradation. Regulation of PTHrP levels is dependent on VDR expression, as using siRNAs to deplete VDR expression negates the 1,25D-mediated downregulation of PTHrP expression. These results indicate the importance of maintaining adequate 1,25D levels and VDR status to control PTHrP levels.

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDRE hPTHrP) within the human PTHrP gene and involves an interaction between nVDRE hPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRα forms part of the nuclear protein complex that interacts with nVDRE hPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9- cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9- cis-retinoic acid enhances the 1,25(OH) 2D 3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9- cis-retinoic acid. Promoter activity driven by nVDRE hPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH) 2D 3 and EB1089 in both cell lines. Co-treatment with these compounds and 9- cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH) 2D 3 in a cell type-specific manner in prostate cancer cells.

Parathyroid hormone-related protein production in the lamprey Geotria australis: developmental and evolutionary perspectives

This study explored the distribution of parathyroid hormone-related protein (PTHrP) and its mRNA in tissues of the lamprey Geotria australis, a representative of one of the two surviving groups of an early and jawless stage in vertebrate evolution. For this purpose, antibodies to N-terminal and mid-molecule human PTHrP were used to determine the locations of the antigen. Sites of mRNA production were demonstrated by in situ hybridisation with a digoxigenin-labelled riboprobe to exon VI of the human PTHrP gene. The results revealed that antigen and its mRNA were widely distributed among similar sites of tissue localisation to those described for mammalian and avian species. However, some novel sites of localisation, such as in the gill and notochord, were also found. Some differences in PTHrP localisation were noted among individuals at different intervals of the life cycle, indicating that the distributions of PTHrP, and possibly its roles, change with the stage of development in this species. The widespread tissue distribution in G. australis implies diverse physiological roles for this protein. The presence of PTHrP in the lamprey, a representative of a group of vertebrates, which apparently evolved over 540 million years ago, strongly suggests that it is a protein of ancient origin. In addition, the successful use of antibodies and probes based on the human sequence in the lamprey also provides evidence that the PTHrP molecule may have been conserved from lampreys through to humans.

Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability.

Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3'-untranslated region (3'-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-beta1 (TGF-beta1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-beta1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-beta1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein-RNA binding studies identified different proteins binding to the 3'-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-beta1-induced changes in PTHrP mRNA isoform expression and stability.

Quantitative evaluation of alternative promoter usage and 3' splice variants for parathyroid hormone-related protein by real-time reverse transcription-PCR.

Parathyroid hormone-related protein (PTHrP) was originally isolated from specific cancers as the primary cause of humoral hypercalcemia of malignancy, a paraneoplastic syndrome occurring in humans with a wide variety of malignancies (1). PTHrP also has been reported to be overexpressed by many types of neoplasms not associated with hypercalcemia (2). PTHrP is a polypeptide hormone with structural similarities to parathyroid hormone (PTH) (3)(4). Amino-terminal fragments of PTHrP exert PTH-like actions in bone and kidney by binding to a common receptor for PTH/PTHrP (PTH1 receptor), producing hypercalcemia (5)(6)(7)(8). High expression of PTHrP by cancer cells also has been proposed to play a role in the progression of breast cancer metastasis to bone (9)(10)(11). The human PTHrP gene is composed of nine exons (Fig. 1A⇓ ). Products of exons 5 and 6 are present in all PTHrP transcripts and encode for the prepro region and the majority of the mature peptide. Alternative splicing of the 3′ end produces three PTHrP isoforms 139-, 173-, and 141-amino acids in length. Transcriptional regulation of the PTHrP gene is achieved by three distinct promoters located at the 5′ end and identified as P1, P2, and P3, respectively. Alternative promoter usage has been evaluated previously by reverse transcription (RT)-PCR based on 5′ alternative splicing, and previous studies showed that P3-initiated transcripts were detectable in most tumors, whereas transcripts initiated by either P1 or P2 were present in only a subset of tumors (12)(13)(14)(15)(16). We describe a novel real-time RT-PCR assay for the specific quantification and characterization of PTHrP mRNA expression, alternative promoter usage, and 3′ splicing in a variety of cancer cell lines as well as healthy and neoplastic human lung tissues. Neoplastic and …

Prostate cancer cell type-specific regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases the growth and enhances the osteolytic effects of prostate cancer cells, it is important to control the level of PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and its non-calcemic analogue, EB1089, suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines PC-3 and LNCaP. The human PTHrP gene contains a sequence element homologous to the negative vitamin D response element within the parathyroid hormone gene. This DNA sequence (nVDRE hPTHrP) bound the vitamin D receptor (VDR) present in nuclear extracts from both PC-3 and LNCaP cells. However, when cloned upstream of the SV40 promoter and transiently transfected into PC-3 and LNCaP cells, nVDRE hPTHrP downregulated promoter activity in response to 1,25(OH) 2D 3 or EB1089 treatment in LNCaP, but not in PC-3, cells. These results may help to explain why some prostate cancers appear to be refractory to treatment with vitamin D analogues.

Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchs.

This study describes the distribution of parathyroid hormone-related protein (PTHrP) antigen and its mRNA in seven species of cartilaginous fish from six elasmobranch families. Antigen was detected using antibodies to synthetic human PTHrP and the mRNA with a riboprobe to human PTHrP gene sequence. The distribution pattern of PTHrP in the cartilaginous fish studied, reflected that observed in mammals but PTHrP further occurs in some sites unique to cartilaginous fish. Of particular note was the demonstration of PTHrP in the shark skeleton, which although considered not to contain bone, may form by a process similar to that forming the early stages of mammalian endochondral bone. The distribution of PTHrP in the elasmobranch skeleton resembled the distribution of PTHrP in the developing mammalian skeleton. Differences in the staining pattern between antisera to N-terminal PTHrP and mid-molecule PTHrP in the brain and pituitary suggested that the PTHrP molecule might be post-translationally processed in these tissues. The successful use of antibodies and a probe to human PTHrP in tissues from the early vertebrates examined in this study suggests that the PTHrP molecule is conserved from elasmobranchs to humans.

Parathyroid hormone-related protein (PTHrP) in cartilaginous and bony fish tissues

Tissues from a range of fish were examined for the presence of parathyroid hormone-related protein (PTHrP) to investigate PTHrP protein distribution and PTHrP gene expression in jawless fish, cartilaginous fish, and bony fish. Immunoreactive PTHrP was localized using antisera to N-terminal and mid-molecule regions of human PTHrP and PTHrP gene expression examined using a digoxigenin labeled riboprobe to a conserved region of the mammalian PTHrP gene. In all of the fish studied, PTHrP protein and messenger RNA (mRNA) were localized to the skin, kidney, and skeletal muscle, following the pattern seen in higher vertebrates. Additional sites of localization for both protein and mRNA included gill, nerve cord, and pituitary, as well as developing dermal denticles and rectal gland in the elasmobranch species. The sites of PTHrP distribution indicate that PTHrP may have roles in ionoregulation as well as growth and differentiation in fish, as has been suggested in higher vertebrates. The results imply that the distribution of PTHrP is widespread in fish and that there is homology between the PTHrP molecules found in humans and fish. The conservation of localization and possible similarity of the PTHrP molecules between tetrapods and fish suggests that PTHrP has a number of fundamental roles in vertebrates. J. Exp. Zool. 284:541–548, 1999. © 1999 Wiley-Liss, Inc.

Characterization of the cell-specific expression of parathyroid hormone–related protein in normal and neoplastic prostate tissue

Objectives. Parathyroid hormone–related protein (PTHrP) is a primary factor in the pathogenesis of malignancy-associated hypercalcemia. By alternative splicing, the human PTHrP gene can generate three different species of mRNA that encode three initial translational isoforms of 139, 173, and 141 amino acids. We recently reported that PTHrP was present in normal prostatic neuroendocrine cells and was overexpressed in prostate cancer tissue as demonstrated by immunostaining. This study was undertaken to further clarify the complex expression of PTHrP gene in normal prostate tissue and prostate cancer. Methods. PTHrP mRNA in samples prepared from normal prostate tissue, prostate cancer, and three prostate cancer cell lines, PC3, LNCaP, and DU145 was assessed using Northern hybridization. Expressed PTHrP isoforms were deduced from differential reverse transcription-polymerase chain reaction (RT-PCR) assays with exon-specific primers. Further localization of different species of PTHrP mRNA was performed using nonradioactive in situ hybridization with exon-specific probes on consecutive sections of normal and neoplastic prostate tissue. Results. Northern hybridization showed that the PTHrP expression level was higher in prostate cancer than in normal prostate tissue. All three PTHrP isoforms could be detected in normal prostate tissues and prostate cancer with differential RT-PCR. Further analysis using in situ hybridization with exon-specific probes revealed that all three PTHrP isoforms were present in prostatic neuroendocrine cells and only PTHrP-1–139 isoform could be clearly detected in prostate cancer tissue. Two androgen-insensitive cell lines, PC3 and DU145, derived from a bone metastasis and a brain metastasis, respectively, expressed all three mRNA species encoding for the three isoforms, but DU145 cells expressed less than PC3 cells. Androgen-sensitive LNCaP cells exhibited a low level of expression of mRNA species encoding for PTHrP-1–139 and PTHrP-1–173, and no expression of PTHrP1-141 isoform. Conclusions. All three initial translational isoforms of PTHrP are produced by prostatic neuroendocrine cells. The mature products of PTHrP might exert their effects on other prostatic epithelial cells in a paracrine fashion and also participate in the homeostatic regulation of the ejaculate. In prostate cancer, differential expression of these three isoforms is evident and PTHrP-1–139 isoform is more abundant than the other two forms. These findings are valuable for designing future research studies to further elucidate the biological functions of PTHrP in normal prostatic glands and prostate cancer.

Differential regulation of the parathyroid hormone-related protein gene P1 and P3 promoters by cAMP

The role of calcitonin, and other agonists which activate the cAMP pathway, in regulating transcription of the human parathyroid hormone-related protein (PTHrP) gene was investigated in a human lung cancer cell line (BEN). Both calcitonin and forskolin caused a 5–6-fold increase in transcription initiated from both the P1 and P3 promoters, but with no observed effect on the P2 promoter. Maximal 6-fold activation of the P1 promoter occurred at 16 h post-stimulation and effects of calcitonin were observed within the pM range. The PKC agonist, phorbol 12-myristate 13-acetate diester (PMA), did not modulate transcription initiated from the P1 promoter. The ionophore ionomycin had a small effect on transcription of the P1 promoter, and transcriptional control may involve an interaction between the cAMP and intracellular calcium second messenger pathways. Deletion mapping studies indicated that increases in transcription of the human PTHrP gene is being mediated via a CRE element situated at −3313 to −3306 upstream of the P1 promoter. Mutational analysis of this CRE element confirmed a role for this sequence in mediating the increase in transcription effected by cAMP. Consistent with these transfection studies, RT-PCR of PTHrP mRNA also indicated a significant increase in transcripts generated from the P1 promoter. Gel retardation assays utilising a fragment of the P1 promoter region, encompassing the putative CRE, determined that nuclear proteins were binding to this region. Competition binding studies with labelled probe and cold competitors determined that the binding was specific for this sequence. A wild-type CRE consensus oligonucleotide also competed for binding with this sequence.

Parathyroid hormone-related protein: evidence for isoform- and tissue-specific posttranslational processing.

Parathyroid hormone-related protein (PTHrP) is expressed by malignant tumors and leads to the syndrome of humoral hypercalcemia of malignancy. It is also expressed by a wide variety of nonmalignant tissues, in which it appears to play distinct paracrine and/or autocrine roles. The human PTHrP gene encodes three cDNA-predicted initial translational products of 139, 141, and 173 amino acids. Most human cell lines contain mRNAs encoding all three PTHrP isoforms. The physiological rationale for the existence of these three highly similar transcripts is unknown. In order to determine whether the protein products derived from these three transcripts differ, we transfected Chinese hamster ovary (CHO) cells and rat insulinoma (RIN) cells individually with cDNAs encoding human PTHrP(1-139), PTHrP(1-141), and PTHrP(1-173). Cell extracts and conditioned medium were then chromatographed using reversed-phase HPLC and analyzed using region-specific PTHrP immunoassays. As we had previously observed in SKRC-1 (renal cell carcinoma) and RIN(1-141) cells, multiple amino-terminal PTHrP species as well as a separate midregion PTHrP species were identified in all six cell lines. In addition, both CHO and RIN cell lines transfected with the PTHrP(1-139) construct contained a previously unrecognized carboxy-terminal fragment that reacted with a PTHrP(109-138) antiserum. This carboxy-terminal fragment was physically distinct from the midregion fragment discovered earlier and was also present in conditioned medium, indicating that it is a secretory form, rather than a biosynthetic intermediate or a degradation product.(ABSTRACT TRUNCATED AT 250 WORDS)

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, 5α-dihydroxy-testosterone 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. Key words: Dexamethasone; Parathyroid hormone-related protein; Gene regulation

Calcitonin increases transcription of parathyroid hormone-related protein via cAMP

Transcriptional regulation of the human parathyroid hormone-related protein (PTHrP) gene by calcitonin was examined in a lung cancer line (BEN cells). Northern analysis demonstrated that calcitonin caused a rapid 4.5-fold elevation in PTHrP mRNA. Transient transfection of a construct containing 1119 base pairs of the human PTHrP gene 5′ to the ATG start site of translation, fused to the CAT reporter sequence, was used to demonstrate a five-fold increase in transcription by calcitonin. Similar increases were also observed when transfected cells were exposed to a number of cAMP agonists including forskolin, as well as isobutyl-methylxanthine. A putative cAMP responsive element (5′-TGACTTCA-3′) present within exon 4 was placed upstream of the heterologous SV40 promoter. Expression of this construct was elevated 4.5-fold in response to calcitonin and 7-fold in response to forskolin. Similar responses to calcitonin occurred with a smaller construct (pZMR30) containing 530 bp of sequence upstream of the ATG start site. Thus we postulate that calcitonin acts at least partially via cAMP through this element in exon 4 of the human PTHrP gene.

Transforming growth factor β stimulation of parathyroid hormone-related protein (PTHrP): A paracrine regulator?

The regulation of PTHrP expression and production by transforming growth factor-β (TGFβ) has been investigated in an epidermal squamous cancer cell line COLO 16. TGFβ 1 treatment increased steady-state levels of PTHrP mRNA and concentrations of PTHrP immunoreactivity in conditioned medium in a time- and dose-dependent manner with a half-maximal effect at 40 pM. An effect of TGFβ 1 on PTHrP mRNA was observed first after 4 h treatment and continued to increase up to 48 h with a concomitant increase in PTHrP immunoreactivity in the culture medium. TGFβ 1 was found to stabilize PTHrP mRNA as assessed by actinomycin C 1 experiments. In addition, a direct effect of TGFβ to increase PTHrP transcription was indicated by nuclear run-on and transient transfection experiments using a CAT promoter/expression construct encompassing the region −1100 bp to −20 bp from the initiating AUG of the human PTHrP gene. The conditioned medium from COLO 16 cells was also shown to contain both latent and active TGFβ at concentrations of 160 pM and 16 pM, respectively, in 72 h conditioned medium. A neutralizing antibody to TGFβ 1 (and TGFβ 2) decreased the level of immunoassayable PTHrP in the medium.

Structure of the 5' flanking region of the gene encoding human parathyroid-hormone-related protein (PTHrP)

We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene ( PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene ( PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p 12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at −514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.