Investigation of Actions of Parathyroid Hormone-Related Protein (PTHrP) Independent of PTH1R

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Abstract

Parathyroid hormone-related protein (PTHrP; gene name PTHLH/Pthlh) acts as a paracrine/autocrine factor to produce a range of effects, including contributing to skeletal development and determining trabecular bone mass. By activating the receptor (PTH1R) that PTHrP shares with parathyroid hormone (PTH), it transduces cascades coupled with the stimulation of cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) and CREB responses. However, in addition to its amino-terminal region that mediates similar actions to PTH, PTHrP has structural features that allow it to exert distinct activities independent of PTH1R. Relatively little is known about the signalling pathways that guide these actions but the novel specific functional regions that exist in PTHrP allow us to ask how PTHrP acts independently and uses non-canonical pathways. The major aim in my thesis is to unveil the PTHrP functions that act through non-canonical pathways associated with regions of the molecule other than PTH1R binding region.

Recently, it has been demonstrated that PTHrP overexpression drives dormant human MCF7 breast cancer cells in mice to colonise the bone marrow and induces osteolytic damages. However, I found that although MCF7 cells activate cAMP in response to prostaglandin E₂ (PGE₂) and calcitonin, the cells do not respond to PTHrP or PTH. This suggests PTH1R in those cells is not functionally linked to adenylyl cyclase. My analysis suggests MCF7 cells express PTH1R mRNA and proteins but such PTH1R does not bind sufficient exogenous PTHrP to activate cAMP. RNA-Seq analysis identified many genes induced by PTHrP overexpression in MCF7 cells, indicating responsiveness to PTHrP, although a shift in cell differentiation because of clonal drift cannot be ruled out. Several potential alternative signalling pathways were identified, notably those related to calcium signalling. These data suggest that the effect of PTHrP overexpression on MCF7 cell dormancy may occur through PTH1R-independent actions of PTHrP.

PTHrP is also essential for bone formation, as indicated by mouse models with genetic depletion of PTHrP, both globally and within the osteoblast lineage. It has been established that Dmp1Cre.Pthlh^f/f mice have significantly lowered levels of PTHrP in osteocytes resulting in reduced bone mass and strength. This differs strikingly from Dmp1Cre.Pth1r^f/f mice with a deficiency of PTH1R in osteocytes which exhibit high bone mass. This implies that PTHrP signalling in osteocytes that promotes bone formation occurs through a non-PTH1R-mediated pathway. To identify alternative signalling pathways of PTHrP, RNA-Seq was conducted on OCY454 osteocytes overexpressing secreted PTHrP (OCY454 PTHrP^FL) and mutant forms of PTHrP lacking both the nuclear localisation sequence (NLS) and C-terminus (OCY454 PTHrP^ΔNLSΔC), the nuclear localisation sequence (OCY454 PTHrP_ΔNLS), or lacking the signalling peptide required for secretion (OCY454 PTHrP_ΔSec). In these cells, expression of PTHrP-activated genes is significantly higher when the PTHrP NLS and C-terminus domains are absent. This shows that the NLS and/or the C-terminus of PTHrP may have a negative impact on genes regulated through PTH1R. However, unlike deletion of the NLS and C-terminus, deletion of NLS alone did not change the number of genes regulated by PTHrP nor the magnitude change. This difference indicates that the C-terminus, but not the NLS, may limit gene expression in response to PTH/PTHrP signalling in OCY454 cells. Importantly, there was no change in cAMP response, CREB responsive gene alterations, or CREB phosphorylation in response to exogenous PTHrP in cells overexpressing the C-terminus, nor was there any difference in the effects of exogenously supplied C-terminus PTHrP compared to full-length PTHrP. This suggests the inhibitory action of the PTHrP C-terminus is intracrine, with regulation taking place beyond CREB activation.

I next performed studies to determine whether the PTHrP C-terminus reduces PTH1R-mediated signalling using qPCR on UMR106.01 overexpressing the C-terminus, focusing on the marked down-regulation of osteocalcin (protein name: BGLAP, bone gamma carboxyglutamate protein; gene name: Bglap1/2) expression, as an example, targeted by PTH/PTHrP signalling. The induction of Bglap1/2 expression by PTH or PTHrP was significantly lower in osteoblastic-like osteosarcoma UMR106.01 cells overexpressing the PTHrP C-terminus. Subsequent analysis suggests this C-terminus gene suppression effect is cytosolic, but is not dependent on modifying cAMP/PKA signalling. My preliminary data suggests modification of Wnt signalling pathway may be required for this C-terminus effect.

The RNAseq data from OCY454 PTHrP^FL cells revealed that osteocyte-derived PTHrP may regulate mineralisation due to significant regulation of mineralisation genes in the gene profile, compared to vector control cells. Since bones from 12-week-old Dmp1Cre.Pthlh^f/f mice were previously reported to have impaired bone material strength, I next sought to understand how osteocyte-derived PTHrP modifies mineralisation in vivo. I used high resolution synchrotron-based Fourier Transform Infrared (FTIR) microspectroscopy to determine the bone composition of 12-week-old Dmp1Cre.Pthlh^f/f mice and Dmp1Cre controls. Male Dmp1Cre.Pthlh^f/f mice showed significantly higher amide I:II ratio (i.e. less compacted collagen) and lower mineral:matrix ratio compared with controls. This suggests a requirement of PTHrP for both collagen organisation and mineral formation. Surprisingly, preliminary in vitro results showed that both PTHrP knocked-down cells and overexpression cells had a low mineralised deposit phenotype. My preliminary data suggests that PTHrP is likely to contribute to bone strength via proper mineral deposition and compaction of collagen, and by modifying osteocyte differentiation.

In conclusion, my thesis describes three major findings. Firstly, although PTHrP promotes breast tumour MCF7 cells aggressively growing in bone, this is not dependent on PTH1R/cAMP/PKA activation. Secondly, in osteocytes, which express PTHR1, the PTHrP C-terminus appears to inhibit Bglap1/2 transcription induced by PTH/PTHrP and this occurs through intracellular pathways independent of PTH1R/cAMP/PKA. Moreover, PTHrP is essential for normal bone composition. It is likely that PTHrP contributes to the promotion of mineralisation, thereby contributing to bone strength via collagen compaction, and by modifying osteocyte differentiation. Deciphering these actions above and their crosstalk with non-PTH1R-mediated processes will increase our understanding of how PTHrP manipulates breast cancer metastasis and bone composition.

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