Biochemical Pharmacology 169 (2019) 113627

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Brief exposure to full length parathyroid hormone-related protein (PTHrP) T

causes persistent generation of cyclic AMP through an endocytosis-
dependent mechanism
⁎
Patricia W.M. Hoa, Audrey S. Chana,c, Nathan J. Pavlosc, Natalie A. Simsa,b, T. John Martina,b,
a
Bone Biology and Disease Unit, St. Vincent’s Institute of Medical Research, Melbourne, Victoria 3065, Australia
b
Department of Medicine, The University of Melbourne, St. Vincent’s Hospital, Melbourne, Victoria 3065, Australia
c
School of Biomedical Sciences, The University of Western Australia, Nedlands, Western Australia 6009, Australia

A R T I C LE I N FO A B S T R A C T

Keywords: Parathyroid hormone (PTH)-related protein (PTHrP) (gene name Pthlh) was discovered as the factor responsible
PTH for the humoral hypercalcemia of malignancy. It shares such sequence similarity with PTH in the amino-terminal
PTHrP region that the two are equally able to act through a single G protein-coupled receptor, PTH1R. A number of
Adenylyl cyclase biological activities are ascribed to domains of PTHrP beyond the amino-terminal domain. PTH functions as a
Receptor
circulating hormone, but PTHrP is generated locally in many tissues including bone, where it acts as a paracrine
Endocytosis
factor on osteoblasts and osteocytes. The present study compares how PTH and PTHrP influence cyclic AMP
(cAMP) formation through adenylyl cyclase, the first event in cell activation through PTH1R. Brief exposure to
full length PTHrP(1–141) in several osteoblastic cell culture systems was followed by sustained adenylyl cyclase
activity for more than an hour after ligand washout. This effect was dose-dependent and was not found with
shorter PTHrP or PTH peptides even though they were fully able to activate adenylyl cyclase with acute
treatment. The persistent activation response to PTHrP(1–141) was seen also with later events in the cAMP/PKA
pathway, including persistent activation of CRE-luciferase and sustained regulation of several CREB-responsive
mRNAs, up to 24 h after the initial exposure. Pharmacologic blockade of endocytosis prevented the persistent
activation of cAMP and gene responses.
We conclude that full length PTHrP, the likely local physiological effector in bone, differs in intracellular
action to PTH by undergoing endosomal translocation to induce a prolonged adenylyl cyclase activation in its
target cells.

1. Introduction
PTH and the autocrine/paracrine factor PTHrP share such structural
similarities within their amino-terminal domains that they act with
equal potency on a common G protein-coupled receptor (GPCR), the
parathyroid hormone receptor (PTH1R) [1–4] (gene name: Pth1r). Li-
gand-induced activation of PTH1R activates adenylyl cyclase [5], with
the generated cAMP responsible for activation of protein kinase A
(PKA) [6] followed by phosphorylation of the cyclic AMP response
element binding-protein (CREB) and activation of downstream tran-
scriptional targets [7,8].
Results of genetic ablation of Pth1r in cells or in mice, or of receptor
antagonism are often used to reach conclusions about PTH action
[9–14], even though PTHrP is an equally effective PTH1R agonist. The
GPCR-mediated action of PTH and PTHrP is unusual in that although
these two alternative agonists have sufficient sequence identity in their
N-terminus for equal recognition by the receptor, they have different
physiological roles. PTH acts as a circulating hormone to regulate ex-
tracellular calcium homeostasis, with its plasma levels subject to ne-
gative feedback control by the prevailing plasma calcium level [15],
whereas PTHrP is produced in many tissues and acts locally as a
paracrine factor [16,17]. This is evident from mouse genetic studies
revealing that PTHrP of osteoblast [18] and osteocyte origin [19] is
necessary postnatally for normal bone remodeling and for maintenance
of bone mass and bone strength. In contrast PTH is required in the fetus
for osteoprogenitor recruitment and bone mineralization, but changes
its function at birth to act on bone primarily to cause bone resorption
and contribute to calcium homeostasis [20–22].
For each of PTH and PTHrP, the full capacity to activate adenylyl
cyclase is contained within the first 34 amino acids [2–4,23]. However,

⁎
Corresponding author at: Bone Biology & Disease Unit, St. Vincent’s Institute of Medical Research, 9 Princes St, Fitzroy, VIC 3065, Australia.
E-mail address: jmartin@svi.edu.au (T.J. Martin).

https://doi.org/10.1016/j.bcp.2019.113627
Received 4 July 2019; Accepted 28 August 2019
Available online 30 August 2019
0006-2952/ Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.
P.W.M. Ho, et al.
at least in the case of PTHrP, the remainder of the molecule also has
significant effects on cellular function [24]. For example, the mid-mo-
lecular domain of PTHrP promotes placental calcium transport [23,25],
and many cellular actions have been identified in response to PTHrP
(107–139) and PTHrP(107–111) [26–30]. In addition, a nuclear loca-
lizing sequence is contained within PTHrP(67–94) [31,32] enabling its
transport into the nucleus by importin β [33]. Despite much effort, no
novel receptor for the carboxy-terminal region has been identified, and
whether the C-terminal portion of PTHrP modifies the initial activation
of PTHR1 remains unresolved. Our interest in whether the circulating
hormone, PTH, and the paracrine factor, PTHrP, differ in their actions
upon PTH1R in target cells is prompted further by our finding in the
immortalized osteocyte cell line, Ocy454 [19] that the full length mo-
lecule PTHrP(1–141) was the only secreted form of PTHrP capable of
acting upon the PTH1R [19].
Although full length PTHrP(1–141) is the form that is produced
locally, our knowledge of effects resulting from PTH1R action on os-
teoblasts is limited to studies of N-terminal PTH and PTHrP (e.g.
[34–39]). Those few exceptions that have used PTHrP(1–141) [4,19,40]
have not studied PTH1R-mediated intracellular signalling duration. In
earlier work in which we used transcriptional array to determine
whether PTH(1–34) and PTHrP(1–141) differed in the patterns of gene
expression that they induced in osteoblasts, no discernable differences
between the two were found [40]. In the present work, we have sought
to determine whether the full-length physiological form of PTHrP dif-
fers in early action from that of the more commonly studied N-terminal
portions of PTHrP and PTH. We also compare these proteins with
abaloparatide, a synthetic peptide recently approved for osteoporosis
therapy that is based on PTHrP and PTH N-terminal sequences [41,42].
We have addressed this question in the present experiments, using
osteoblast, osteocyte and osteosarcoma cell lines and primary mouse
calvarial osteoblasts, by investigating whether brief exposure to PTH
and PTHrP preparations, followed by wash-out of treatments, influ-
ences their efficacies in inducing persistent activation of adenylyl cy-
clase. The latter has been assessed at intervals after wash-out by mea-
suring cAMP formation in the presence of maximum phosphodiesterase
inhibition [43–45]. Later cellular events investigated include activation
of CREB, and the expression of genes responsive to this pathway. Fur-
thermore, since receptor endocytosis and subsequent delivery to en-
dosomes is increasingly recognized as a mechanism that results in
sustained intracellular signals [43,46,47], we have examined that
possibility using a pharmacological approach to block inhibitors of
endocytosis.
2. Materials and methods
2.1. PTHrP and PTH preparations
Recombinant human PTHrP(1–141), (1–108) and (1–84) were pre-
pared by expression in E. Coli as previously described [4]. Human PTH
(1–34), human PTHrP(1–34), and human PTHrP(1–36) were purchased
from GL Biochem Ltd (Shanghai, China); human PTH(1–84) was pur-
chased from Bachem (Bubendorf, Switzerland). Abaloparatide from two
sources was used with identical outcomes, synthesized by Bachem
(Bubendorf, Switzerland) and GL Biochem Ltd (Shanghai, China). M-
PTH(1–34) [48] was provided by Dr T Gardella (Massachussetts Gen-
eral Hospital). Mass spectrometry was used to establish purity of all
peptide preparations. Forskolin was purchased from Sigma-Aldrich
(Castle Hill, Australia).
2.2. Dynamin inhibitors
The dynamin inhibitor Dyngo4a was purchased from Abcam
(Melbourne, Australia) and the endocytosis inhibitor Pitstop2 from
Sigma-Aldrich. Each was stored as a 30 mM stock in DMSO (Sigma-
Aldrich) and diluted appropriately for use in cells at less than 1% final
2
Biochemical Pharmacology 169 (2019) 113627
DMSO concentration.
2.3. Cell culture
Mouse calvarial osteoblasts were prepared by enzymatic digestion
of bones from 1- to 3-day-old C57BL/6 pups as previously described
[49,50]. Rat (UMR106-01) [51] and mouse (494H) osteogenic sarcoma
cells [52], and MC3T3-E1 cells were cultured as previously described.
Human embryonic kidney (HEK293) cells were generated to over-
express amino terminal-tagged GFP-PTH1R using Lipofectamine LTX
(Thermo Fisher, Australia) and maintained in G418 (500 µg/mL, Life
Technologies, Australia) antibiotic selection, as described in [8].
2.4. Cyclic AMP generation
The maximum cAMP response to each agonist was measured by
assaying cAMP after acute treatment for 12 min in the presence of 1 mM
isobutylmethylxanthine (IBMX, Sigma Aldrich) as phosphodiesterase
inhibitor. In persistent activation experiments, cells were treated in 12-
well plates with indicated ligands using 0.1% BSA in Alpha MEM as
assay buffer and incubated at 37 °C, 5% CO2 for 12 min. Assay medium
was then aspirated, cells rinsed twice with 1 ml PBS at room tempera-
ture and washes removed with vacuum pump. Fresh assay medium at
37 °C was then added and cells were incubated for a further 30, 60, 90
and 120 min, at which times IBMX (1 mM) was added for the last
12 min. Reaction was terminated with 1.5 ml acid ethanol immediately
after aspiration of medium. Extracts were dried and reconstituted in
assay buffer for cAMP radioimmunoassay as described [45]. This pro-
cedure allows a measure of the state of activation of adenylyl cyclase at
that time [43,45,48,52].
2.5. Transfections and luciferase assays
UMR106-01 cells were stably transfected with pCRE-luciferase
(Clontech, California, USA), with a vector containing multiple copies of
the CRE binding sequence fused to a TATA-like promoter region from
the Herpes simplex virus thymidine kinase promoter and an ampicillin-
resistance gene for selection [53]. Selection of stable clones was with
G418 (1 mg/ml). Luciferase activity was quantitated using the Polarstar
Optima, with substrate from Promega Australia. The Invitrogen RNAi
design tool was used to select siRNA duplex sequence for rat protein
kinase inhibitor γ (PKIγ). The siRNA sequence selected was, GAG ACA
UGG GCG AGC UCG CAC UUG A.
2.6. Real time PCR
RNA was extracted with TRIzol (Thermo Fisher Scientific,
Massachusetts, USA) and treated with DNase (Ambion, Austin, TX,
USA) according to the manufacturer’s instructions. RNA was quantified
by measuring absorbance at 260 nm. cDNA was prepared from RNA
using random primers (Invitrogen) and Superscript III (Thermo Fisher
Scientific, Massachusetts, USA) as previously described [45,54]. Real-
time RT-PCR was performed using Sybr Green (Applied Biosystems,
Australia) on an MX3000P plate reader (Stratagene, La Jolla, CA). Data
were normalized to hypoxanthine-guanine phosphoribosyl transferase
(Hprt1) and represented as fold induction over untreated time-point
control. Real-time PCR data were analyzed using Mx-Pro QPCR soft-
ware (Stratagene). Primers were manufactured by Integrated DNA
Technologies (IDT Australia, Boronia) and were as previously de-
scribed: murine Tnfsf11 (RANKL), Fos and Hprt1 [55], Il6, Il11 [56],
Rgs2, Bglap1, Sost, Hprt1 and Efnb2; rat Tnfsf11 [40], Sost [57], Nr4a2
[45], Il6 [58]. Rat Bglap1 primers were as follows: forward: 5′-CCGG
AGTCTATTCACCACCT-3′, reverse: 5′-GACAAGTCCCACACAGCAAC-3′,
rat Nr4a2 were forward: 5′-AGATTCCTGGCTTTGCTGAC-3′, reverse: 5′-
CTGGGTTGGACCTGTATGCT-3′, and rat Pkig primers as described in
[59].
P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

Fig. 1. Persistent cAMP formation after short term treatment with full-length PTHrP. A,B: Duration of cAMP formation in UMR106-01 cells incubated for 12 min with
PTHrP(1–34), PTHrP(1–84), PTHrP(1–108), PTHrP(1–141), PTH(1–34), PTH(1–84), or Forskolin (all at 100 nM) followed by wash-out and replacement with
peptide-free medium, incubated for the times indicated with 1 mM IBMX added for the last 12 min. All results are expressed as percent of maximum cAMP levels
generated with each peptide at 100 nM for 12 min; maximum cAMP levels are as follows (pmol/wel): PTHrP(1–34), 364 ± 4; PTHrP(1–84), 271.5 ± 24.2, PTHrP
(1–108), 322.2 ± 17.0; PTHrP(1–141), 319.7 ± 4.9; PTH(1–34), 312.3 ± 5.1; Forskolin, 232.2 ± 5.5. C: Duration of cAMP response with PTHrP(1–141) at 0.1, 1,
10 and 100 nM in preincubation, with washout followed by cAMP formation assay as in panel A; Maximum cAMP levels generated at each PTHrP(1–141) con-
centration are (pmol/well): 100 nM, 345 ± 8.3; 10 nM, 305.5 ± 9.7, 1 nM, 143.9 ± 2.6; 0.1 nM, 10.7 ± 0.9. D: Abaloparatide does not induce persistent cAMP
formation in UMR106-01 cells; protocol as in panel A; maximum cAMP levels generated with each ligand (all at 10 nM) are (pmol/well): Abaloparatide: 903 ± 84;
PTH(1–34): 843 ± 12; PTHrP(1–36): 810.5 ± 36; PTHrP(1–141): 886 ± 32. E-H: Persistent cAMP formation in additional osteoblast and PTH1R-expressing cells,
pre-incubated with peptides and assayed as in A,B. Figure legend for all panels on left; all data shown as percentage maximal cAMP to allow comparison between
different cell types, as follows. E: Murine primary calvarial osteoblasts differentiated for 14 days (maximum cAMP response to each treatment are (pmol/well): PTH
(1–34) 100 nM, 313 ± 29; PTHrP(1–141) 100 nM, 293.3 ± 13.3, PTHrP(1–141) 10 nM, 305.7 ± 17.0; M-PTH (100 nM), 257.2 ± 21.9). F: MC3T3-E1 cells
(maximum cAMP response (pmol/well) are as follows: PTH(1–34) 10 nM = 1250 ± 325; PTHrP(1–141) 10 nM = 1230.5 ± 254; PTHrP(1–141)
100 nM = 1743.8 ± 126). G: Murine osteosarcoma Os494H cells (maximum cAMP responses (pmol/well) are as follows: PTH(1–34) 10 nM 211 ± 13; PTHrP(1–36)
10 nM = 240.3 ± 4.7; PTHrP(1–141) 10 nM = 251.3 ± 9.7; PTHrP(1–141) 100 nM = 249.9 ± 4.0). H: HEK293 cells overexpressing PTH1R (maximum cAMP
responses (pmol/well) are as follows (all at 10 nM): PTHrP(1–34) = 454 ± 63; PTHrP(1–141) = 536.1 ± 50; PTHrP(1–34) = 458.2 ± 45.8; M-PTH
(1–34) = 423.5 ± 50.1). Forskolin (at 100 nM) was used as a positive control for cAMP stimulation in all experiments. All data shown in mean ± SEM for n = 3
replicate wells; representative of at least 3 independent experiments.

3. Results The sustained cAMP response to PTHrP(1–141) after peptide wash-

3.1. Prolonged activation of cyclic AMP formation by PTHrP(1–141) after
a brief exposure and wash-out
UMR106-01 cells exposed to PTH(1–34) for 12 min showed a sub-
stantial cAMP response in the presence of IBMX (Fig. 1A,B). This was
not retained after wash-out of the peptide; at all time points after wash-
out, formation of cAMP was < 10% of maximum (Fig. 1A,B). However,
UMR106-01 cells exposed to PTHrP(1–141) retained the ability to
generate a cyclic AMP response in the presence of IBMX even two hours
after wash-out (Fig. 1B); the magnitude of this response was not sig-
nificantly different from the immediate response to maximum stimu-
lation with the same protein. When shorter peptides were tested in the
same cells, a lesser persistent response was obtained following pre-
incubation with PTHrP(1–108) (Fig. 1B), but no persistent effect was
seen after treatment with PTHrP(1–84) (Fig. 1B), nor after exposure to
PTH(1–34) (Fig. 1B), to PTH(1–84) (Fig. 1B), or to the adenylyl cyclase
stimulator, forskolin (Fig. 1B).
out in UMR106-01 cells was dose-dependent (Fig. 1C). Although the
effect was retained at near maximal levels with 100 nM PTHrP(1–141),
treatment with the lower 10 nM dose of PTHrP(1–141) resulted in a
cAMP response of 75% maximum retained 30 min after peptide wash-
out, and remaining at 25% at 60 min; Fig. 1D shows a second experi-
ment with similar results. After wash-out of both 1 nM and 0.1 nM
PTHrP(1–141) a response of 25% maximum was retained at 30 min. We
also tested the actions of abaloparatide, a synthetic ligand, which is
based on the N-termini of PTHrP and PTH, but like other N-terminal
peptides, including PTH(1–34), this did not induce persistent cAMP
formation (Fig. 1D). Because prolonged receptor activation has been
documented with the synthetic peptide M-PTH(1–34), this was also
tested, and it induced persistent activation to the same extent as the
native protein PTHrP(1–141) after brief incubation and wash-out in
primary calvarial osteoblasts (Fig. 1E).
This persistent activation of cAMP formation after washout of
PTHrP(1–141) was confirmed at 30 and 60 min in primary murine
calvarial osteoblasts (Fig. 1E); as in UMR106-01 cells, PTH(1–34)

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P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

Fig. 2. Persistent cyclic AMP response element (CRE) activity in response to PTHrP(1–141) and M-PTH, and effect of PKIγ siRNA. A: Effect of continuous treatment
on cAMP responsive element (CRE)-luciferase signal in stably transfected UMR106-06 cells with peptides as indicated (all at 10 nM). B: Duration of CRE-luciferase
response after 12 min treatment (all at 10 nM) followed by wash-out and assay at indicated times in UMR106-01 cells; legend in panel A. All data shown in
mean ± SEM for n = 3 replicate wells; representative of 3 independent experiments. C: mRNA levels of PKIγ in wild type cells, and in cells transfected with
scrambled siRNA and PKIγ targeted siRNA, cultured for 72 h, showing a significant and stable knockdown of Pkig mRNA; values are mean + SEM; *, p < 0.05 vs
scrambled and wild type cells. D,E: Effect of PKIγ siRNA on CRE-luciferase response after 4 h of continuous treatment (D) or 4 h after 12 min treatment followed by
washout (E): PTH(1–34), PTHrP(1–141) or M-PTH were all used at 10 nM in wild type, scrambled siRNA, and PKIγ siRNA cells. Values are mean + SEM; **,
p < 0.01 vs both wild type and scrambled RNA controls.

treatment did not lead to persistent cAMP formation (Fig. 1E). In ad-
dition, a sustained cAMP response was observed when cells were
treated with the synthetic ligand M-PTH(1–34) [48].
Persistent activation of cAMP formation after wash-out of PTHrP
(1–141) and dose-dependence of that effect was also observed in the
MC3T3-E1 osteoblastic cell line (Fig. 1F) and in OS494H cells (Fig. 1G),
derived from a mouse osteogenic sarcoma in which tumor growth and
invasion is driven by the cAMP/PKA/CREB pathway under the influ-
ence of autocrine/paracrine PTHrP [45,52,60]; no persistent activation
was observed with PTH(1–34) in these cell lines (Fig. 1F,G). In HEK293
cells overexpressing PTH1R (Fig. 1G) dose-dependent persistent acti-
vation of adenylyl cyclase to PTHrP(1–141) was also observed after
wash-out, and M-PTH was at least as effective as full length PTHrP in
the response. Some lesser persistent activation was seen at 60 min post-
washout within PTH(1–34) in these cells that overexpress the PTH1R
(Fig. 1G).
Control experiments were carried out to ensure that PTHrP(1–141)
was not adhering non-specifically to cells. After pre-treatment with
PTHrP(1–141), PTH(1–34) and PTHrP(1–34) and washing, as in the
studies of Fig. 1, cells were treated with glycine buffer, pH 3.0. This
acid eluate was dried and retained for assay. After reconstitution the
acid eluates were found to contain no activity capable of promoting
cyclic AMP formation in the assay cells, nor could any PTHrP be de-
tected by an N-terminally directed radioimmunoassay with a sensitivity
of 2 pM [61].
The overall conclusion from these experiments was that the inter-
action of PTHrP(1–141) with the target cells differed in a major way
from the interactions of its shorter peptides and from PTH(1–34). The
difference was that brief exposure to PTHrP(1–141) followed by wash-
out, resulted in a prolonged ability to generate cAMP in the presence of
phosphodiesterase inhibition, indicating that receptor-adenylyl cyclase
was continuing to signal at a greater rate than in control cells or those
pre-treated with PTH or shorter PTHrP peptides.
3.2. CREB pathway activation is prolonged after brief exposure to PTHrP
(1–141)
Since the studies set out with the premise that the paracrine factor,
PTHrP, might differ in its action from the circulating hormone, PTH, we
next investigated later events in the cell that are related to cyclic AMP
generation. An early event is PKA activation of CREB leading to CREB-
mediated transcription.
We used a CRE-luciferase reporter assay in stably transfected
UMR106-01 cells to examine the duration of effects of 15 min exposure
to PTHrP and PTH peptides followed by wash-out. In previous work
CREB phosphorylation after peptide pre-incubation and washout in
calvarial osteoblasts was found to be prolonged in response to full
length PTHrP compared to (1–108) and (1–84) and of PTH(1–34) [8].

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P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

Fig. 3. Sustained gene expression response to PTHrP(1–141). UMR106-01 cells were treated with PTH(1–34) or PTHrP(1–141) at 10 nM continuously, or incubated
with the peptides for 15 min followed by wash-out and continued incubation in peptide-free medium, then specific mRNAs assayed by qPCR at the indicated times. A:
cFos (Fos) at 1 h. B, C and D: IL-6 (Il6), RGS2 (Rgs2) and ephrinB2 (Efnb2) at 4 h. E, F: RANKL (Tnfsf11) and osteocalcin (Bglap1) at 8 h. G: Sclerostin (Sost) at 24 h.
Forskolin (at 100 nM) was used as a non-PTH1R-dependent stimulus of cAMP in all experiments. Data is mean + SEM. All data shown in mean ± SEM for n = 3
replicate wells; representative of 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs untreated control under the same conditions by one-way
ANOVA.

Continuous treatment with each of PTHrP(1–141), PTHrP(1–34) and
PTH(1–34) resulted in a progressive increase in CRE-luciferase reporter
activity to a maximum at 4 h (Fig. 2A). When cells were treated briefly
followed by wash-out and continued incubation in peptide-free
medium, luciferase activity progressively increased to a maximum at
4 h only when pre-treated with PTHrP(1–141) and M-PTH(1–34)
(Fig. 2B). This CRE-luciferase activation occurred to the same extent
and at the same rate as in continued presence of agonist (Fig. 2B).
Neither PTH(1–34), nor PTHrP(1–34) had this prolonged effect after
peptide wash-out (Fig. 2A,B).
The PKA inhibitor peptide (PKI) has been reported to regulate ter-
mination of PKA signaling, including in osteoblastic cells [6,59,62,63].
Knock-down of PKIγ in osteoblasts delays termination of nuclear PKA
activity and prolongs phosphorylation of CREB and expression of some
early response genes [8,59,63]. Knockdown of PKIγ (Pkig) by siRNA in
UMR106-01 cells (Fig. 2C), did not significantly modify the maximal
CRE-luciferase response at 4 h to continuous treatment with either PTH
(1–34) or PTHrP(1–141) treatment (Fig. 2D). When responses to
treatment with wash-out were tested, the knockdown cells maintained a
greater CRE-luciferase increase 4 h after PTHrP(1–141) or M-PTH
(1–34) pre-treatment (Fig. 2E), as was to be expected with reduction of
the PKA inhibitor. However, the knockdown did not modify the action
of PTH(1–34) on CRE-luciferase activity (Fig. 2E), indicating that the
persistent activation induced by PTHrP(1–141) treatment is unlikely to
be mediated by PKIγ dependent-mechanisms.
3.3. Gene expression in response to either continuous or brief exposure to
agonists.
Since these findings raised the possibility that the sustained effect of
brief exposure to PTHrP(1–141) might influence still later events, we
examined the effect of either continuous or transient treatment with
PTH(1–34) and PTHrP(1–141) followed by wash-out on the expression
of genes known to be influenced by these agonists in UMR106-01 cells
[35,40,64–67]. Continuous treatment with PTH(1–34), PTHrP(1–141),
or forskolin significantly elevated c-fos (Fos) mRNA levels at 1 h
(Fig. 3A). Consistent with the sustained increase in cAMP generation,
exposure to PTHrP(1–141) for 15 min followed by wash-out also led to
an increase in Fos mRNA levels 1 h later (Fig. 3A). The increase was
equivalent to that obtained from continuous treatment for 1 h (ap-
proximately 10-fold); no such extended increase in Fos mRNA was ob-
served with either PTH(1–34) or forskolin after wash-out.
In UMR106-01 cells, 4 h of continuous treatment with PTH or
PTHrP(1–141) is also known to increase mRNA levels for interleukin 6
(Il6), EphrinB2 (Efnb2), and RGS2 (Rgs2) [40,66,68]. This is confirmed
in Fig. 3B-D. After brief exposure and wash-out of PTHrP(1–141), the
same magnitude in elevation of each mRNA was still detected 4 h after
wash-out (Fig. 3 B - D). No extended elevation in these genes was seen
in response to pre-treatment with PTH(1–34) or forskolin. We also as-
sessed genes known to be maximally upregulated at 8 h of continuous
treatment with PTH(1–34) or PTHrP(1–141); the increases in mRNA for
osteocalcin (Bglap1) and receptor activator of NFκB ligand (RANKL)
(Tnfsf11) (Fig. 3E, F); again such increased expression was similar when
cells were exposed to PTHrP(1–141) for only 15 min, and assessed 8 h
after wash-out, but there was no sustained effect of PTH(1–34) or for-
skolin on mRNA levels of these genes (Fig. 3E, F). Finally, mRNA levels
of the bone formation inhibitor, sclerostin (Sost) which is inhibited by
PTH(1–34) or PTHrP(1–141) treatment in these cells through a cAMP/
PKA mechanism [19,69], was confirmed by continuous treatment with
either agonist for 24 h (Fig. 3G). Partial suppression of Sost mRNA le-
vels was detected at 24 h after washout following transient exposure to
PTHrP(1–141) (Fig. 3G). When this experiment was repeated, we

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P.W.M. Ho, et al.
Fig. 4. Repeat of experiment shown in Fig. 3 with M-PTH included to show the similar response to PTHrP(1–141). UMR106-01 cells were treated with PTH(1–34),
PTHrP(1–141) or M-PTH at 10 nM continuously, or incubated with the peptides for 15 min followed by wash-out and continued incubation in peptide-free medium,
then specific mRNAs assayed by qPCR at the indicated times. A: cFos (Fos) at 1 h. B, C and D: RGS2 (Rgs2), IL-6 (Il6), and ephrinB2 (Efnb2) at 4 h. E, F: RANKL
(Tnfsf11) and osteocalcin (Bglap1) at 8 h. Data is mean + SEM, n = 3.
included pre-treatment with M-PTH(1–34), which also causes persistent
activation of adenylyl cyclase (Fig. 1E,H, Fig. 2B). M-PTH(1–34) also
consistently achieved sustained elevations in Fos, Efnb2, Il6, Rgs2,
Bglap1 and Tnfsf11, as observed in response to PTHrP(1–141)
(Fig. 4A–F).
These sustained effects of PTHrP(1–141) on mRNA levels were also
assessed in normal primary osteoblast cultures derived from mouse
calvariae. After establishing calvarial cells and allowing them to dif-
ferentiate in culture for 16–20 days, they were treated either con-
tinuously for the indicated times, or briefly followed by wash-out and
continued culture as above, with each of PTHrP(1–141), PTH(1–34),
PTHrP (1–36) and abaloparatide. Several genes were examined whose
expression responses via PTH1R we could expect from previous data
[40,56,70]. The mRNA levels of each of Tnfsf11, Il6, Il11, and the cAMP
response gene Nr4a2 were all sustained in cultures treated with PTHrP
(1–141) briefly with wash-out, but PTH(1–34) and PTHrP(1–36) in-
creased levels of these genes only if provided continuously (Fig. 5). We
also tested abaloparatide treatment, which appeared to have a very
mild sustained effect on Tnfsf11, but no significant sustained effect on
Nr4a2, Il6 or Il11 (Fig. 5).
6
Biochemical Pharmacology 169 (2019) 113627
3.4. Pharmacologic inhibition of endocytosis blocks persistent cAMP
activation and persistent gene responses induced by PTHrP(1–141)
Since receptor endocytosis and subsequent delivery to endosomes is
increasingly recognized as a mechanism that produces sustained in-
tracellular signals [71], as opposed to long-standing views that cano-
nical GPCR activation and generation of cAMP was confined to the
plasma membrane [43,46,47], we tested whether endocytosis may be
involved in the persistent activation of adenylyl cyclase induced by
PTHrP(1–141). For this purpose, we used Dyngo4a, a small molecule
dynamin inhibitor that can rapidly and reversibly block endocytosis in
minutes [72]. We also used the complementary reagent, Pitstop2,
which acutely interferes with endocytosis [73]. Two inhibitors with
different modes of action were used to minimize off-target effects.
Preincubation of UMR106-01 cells with Pitstop2 for 30 min
(Fig. 6A) had no significant influence on the maximum responsiveness
of cAMP to treatment with PTH(1–34) or PTHrP(1–141) in the presence
of IBMX (Fig. 6B); similarly Dyngo4a generally had only a mild influ-
ence on maximal cAMP response, apart from a 50% inhibition at the
highest dose of 50 μM (Fig. 6A, C); the mechanism of this inhibition is
unknown, but it may relate to impaired resensitization. In cells treated
with Pitstop2 (Fig. 6E) or Dyngo4a (Fig. 6F) and stimulated with pep-
tide agonists for 12 min followed by washout of the cells, the persistent
cAMP accumulation response to pre-treatment with PTHrP(1–141) was
P.W.M. Ho, et al.
dose-dependently and significantly reduced, particularly with Dyngo4a
treatment (Fig. 6F), where inhibition of > 80% was seen with the
lowest Dyngo concentration used (10 μM); a level of inhibition far
greater than that seen with the highest dose of Dyngo4a in continuous
PTHrP(1–141) treatment (Fig. 6C). This indicates that although en-
docytosis is not required for the initial cAMP response to PTH(1–34) or
PTHrP(1–141), it is required for the persistent activation of adenylyl
cyclase induced by PTHrP(1–141).
Since endocytosis appeared to be required for generation of the
persistent cAMP formation response to PTHrP(1–141), we assessed
whether the persistent effects on gene expression were also dependent
on endocytosis, using a similar approach. Three modes of exposing cells
to PTH(1–34) and PTHrP(1–141) were tested and compared to vehicle
(Fig. 7A), including continuous treatment with peptide, treatment fol-
lowed by wash-out, and treatment followed by wash-out after pre-
treatment with Dyngo4a. Both early (Nr4a2 and Il6) and late (Tnfsf11
and Bglap1) cAMP responsive genes were assessed. PTH(1–34) treat-
ment, as previously, increased gene expression significantly when ad-
ministered continuously, but no changes in gene expression were ob-
served after wash-out (Fig. 7B–E). PTHrP(1–141) treatment
significantly increased Nr4a2 and Il6 at 1 h after washout (as in Fig. 2);
the Nr4a2 response was significantly reduced by Dyngo4a pre-treat-
ment, but in this experiment the Il6 response was not. We suggest that
in the case of these rapid early response genes, PKA/CREB- related
events can be initiated too rapidly for any effect of endosomal inhibi-
tion to be clearly shown, although it was shown with Nr4a2. This did
not apply to the later induced genes; Tnfsf11 and Bglap1 mRNA levels
7
Biochemical Pharmacology 169 (2019) 113627
Fig. 5. Effects of PTH(1–34) and PTHrP
(1–141) on gene expression in primary
mouse calvarial osteoblasts allowed to dif-
ferentiate in culture for 14 days to ensure
functional PTH1R expression. After treat-
ment with PTH(1–34) and PTHrP(1–141),
PTHrP(1–34) and Abaloparatide (both at
100 nM) for 15 min followed by wash-out,
mRNAs were assayed by qPCR after 4 hrs
incubation free of treatment. A: RANKL
(Tnfsf11), B: IL-6 (Il6), C: NR4A2 (Nr4a2),
D: IL-11 (Il11). Data is mean + SEM from
triplicate wells; representative of 3 in-
dependent experiments. *, p < 0.05; **,
p < 0.01; ***, p < 0.001 vs untreated
control under the same conditions by one-
way ANOVA.
were increased by PTHrP(1–141) treatment at 8 h after wash-out, and
each response was significantly reduced by Dyngo4a pre-treatment.
This indicates that the sustained gene responses to PTHrP(1–141) target
genes after exposure and wash-out of the protein are, at least in part,
dependent on endocytotic processes.
3.5. PTHrP(1–141) treatment desensitizes cells to PTH1R-induced cAMP
response
To determine whether the sustained cAMP response to PTHrP
(1–141) may result from sustained internalization of the PTH1R, we
assessed PTH1R recovery after PTH(1–34) or PTHrP(1–141) treatment
by assessing cAMP responses to a second exposure given 16 h after in-
itial treatment (Fig. 8A). When cells were pre-treated with PTH(1–34),
there was no diminution in cAMP response to a second exposure, 16 h
after the pre-treatment (Fig. 8B left panel). In contrast, when cells were
exposed to PTHrP(1–141), their cAMP response 16 h after the initial
exposure was significantly reduced by > 50% regardless of whether the
second stimulus was PTHrP(1–141) or PTH(1–34) (Fig. 8B right panel).
This indicates that PTHrP(1–141) treatment desensitizes the cAMP re-
sponse in cells exposed to PTH1R-binding ligands, an effect present
even 16 h after the initial exposure.
We carried out a second experiment to determine whether this effect
of PTHrP is specific to PTH1R-binding ligands, by including pros-
taglandin E2 (PGE2), which activates adenylyl cyclase in these cells, but
does not act through PTH1R [6]. We also assessed earlier time points to
determine at what time the desensitization arises (schematic shown in
P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

A B 500
C 400

400

cAMP (pmol/well)

cAMP (pmol/well)
assay 300
300 * * * *
PTH or PTHrP 200
Pitstop2 or Dyngo4a 200 ***
-30 -12 0 minutes 100
100

0 0
Vehicle 10 30 50μM Vehicle 10 30 50μM Pitstop2 Vehicle 10 30 50μM Vehicle 10 30 50μM Dyngo4a
PTH(1-34) PTHrP(1-141) PTH(1-34) 10nM PTHrP(1-141) 10nM

D E 175 F 150
150
cAMP (pmol/well) * 125

cAMP (pmol/well)
125
wash-out assay 100
100
***
75 75
PTH or PTHrP ***
Pitstop2 or Dyngo4a 50 50
-30 -12 0 60 25 25 ***
minutes *** ***
0 0
Vehicle 10 30 50μM Vehicle 10 30 50μM Pitstop2 Vehicle 10 30 50μM Vehicle 10 30 50μM Dyngo4a
PTH PTHrP(1-141) PTH (1-34) 10nM PTHrP(1-141) 10nM

Fig. 6. Dyngo4a and Pitstop2 effects on persistent activation of adenylyl cyclase. A: Design: test of Dyngo4a and Pitstop2 incubation on maximal cyclic AMP response
to 12 min PTH(1–34) or PTHrP(1–141). B, C: cAMP response of UMR106-01 cells pre-incubated with Pitstop2 (B) or Dyngo4a (dynamin inhibitor, C) for 30 min, prior
to the addition of PTH(1–34) or PTHrP(1–141), at 10 nM for 12 min in the presence of 1 mM IBMX for assay of maximum cyclic AMP generated. D: Design: test of
Dyngo4a and Pitstop2 incubation on cyclic AMP response 60 min after wash-out of PTH(1–34) or PTHrP(1–141). E,F: cAMP response of UMR106-01 cells pre-
incubated with varying concentrations of Dyngo4a or Pitstop2 for 30 min, with the addition of PTH or PTHrP peptides for 12 min before washout and continued
incubation in peptide-free medium. Cyclic AMP was assayed 60 min after 12 min in 1 mM IBMX. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs untreated control
under the same conditions by one-way ANOVA.

Fig. 8C). At no time after PTHrP(1–141) treatment was the cAMP re- prominent feature of PTHrP(1–141) action than it is that of the shorter
sponse to PGE2 reduced (Fig. 8D). In contrast, responses to PTHrP ligands
(1–141) and PTH(1–34) were both impaired after initial exposure to It has been recognized increasingly in the last several years that
PTHrP(1–141). This was observed first at 8 h after treatment, where the signaling through GPCRs can continue after ligand-receptor inter-
response to a second treatment with a PTH1R-binding ligand was re- nalization through endocytosis. Among reported examples of continued
duced to approximately 75%, then to 50% at 16 h (Fig. 8B), and at 24 h, endosomal signaling with sustained cellular responses are those with β-
the cAMP response to both PTH1R-binding ligands was only 25% of adrenergic agonists [75], PTH [76], pituitary adenylyl cyclase acti-
maximum (Fig. 8D). This indicates that PTHrP(1–141) treatment causes vating polypeptide (PACAP) [77], thyroid stimulating hormone (TSH)
a gradual desensitization of cells specifically to PTH1R ligands. We [78], sphingosine-1-phosphate [79], calcitonin gene-related peptide
hypothesized that PTHrP(1–141) may cause a prolonged state of en- (CGRP) [80], and neurokinin 1 [81]. One of the first GPCRs to show
dosomal retention of PTH1R following receptor activation, and that this endosomal signaling was PTH1R, when studies in cells engineered to
may mediate both the persistent activation of cAMP signaling and the overexpress PTH1R obtained differing initial receptor interactions with
subsequent reduced receptor availability at the cell surface PTHrP(1–36) and PTH(1–34). The action of PTHrP(1–36) was restricted
to the cell surface, while PTH(1–34) was more readily internalized to an
4. Discussion endosomal location and brought about a more prolonged increase in
cyclic AMP in the target cells [76]. Receptor interactions similar to
The dominant signaling pathway mediating responses to PTHrP and those with PTHrP(1–36) were also observed with abaloparatide [42],
PTH is the cAMP/PKA pathway, with activation by the amino-terminal which is identical to PTHrP in its first 21 residues, but has 8 residues
domains of either PTH or PTHrP resulting in coupling of PTH1R to Gαs/ different from PTHrP between 22 and 34 [41]. These studies also
adenylyl cyclase/cAMP/protein kinase A (PKA) signaling. In promoting showed prolongation of cAMP response with PTH(1–34) compared with
cAMP formation, PTHrP preparations from amino-terminal to full PTHrP(1–36) and abaloparatide [42], when cAMP was measured in
length have effects equivalent on a molar basis with those of PTH(1–84) cells without phosphodiesterase inhibition. We have used a different
and PTH(1–34) [4,74]. We have used several different target cells and read-out, by measuring cAMP with phosphodiesterase inhibition al-
preparations of PTHrP and PTH of varying lengths, to find consistently lowing assessment of the state of activation of adenylyl cyclase, the first
that PTHrP(1–141) pre-incubation induces a prolonged state of ade- point in the cascade of events mediated through the cAMP/PKA
nylyl cyclase activity in all the cells tested. Substantial but significantly pathway [82]. The present experiments cannot completely exclude the
less activation occurred in our experiments with PTHrP(1–108) but was possibility that shorter peptides have appreciably lesser effects on
not evident with PTHrP(1–34) or (1–36), or with PTH(1–34) or PTH persistent cAMP activation than those with the full PTHrP molecule.
(1–84). We show the persistent adenylyl cyclase activation effect of Such effects could contribute to the net effect on total cellular cAMP
PTHrP(1–141) to be consistent in differentiated calvarial and MC3T3- levels reported in those studies without phosphodiesterase inhbition
E1 osteoblasts, in mouse and rat osteogenic sarcoma cells, and in [42,44,48,76].
HEK293 cells overexpressing PTH1R. This sustained cAMP production Our experiments show that full length PTHrP has a very pronounced
was also reflected in prolonged CRE-luciferase activation and prolonged effect to promote persistent cyclase activation after brief exposure; this
gene expression responses following brief exposure to PTHrP(1–141). was not found with the shorter peptides. Specificity of action within the
Our data indicates that these effects are mediated by an endocytosis- cAMP/PKA pathway requires compartmentalization of cAMP pools and
dependent process. This suggests that receptor internalization is regu- the effector molecules. As examples, phosphodiesterases are required
lated differently by PTHrP(1–141) and PTH(1–34), and is a more for compartmentalization to specific subcellular regions [83], agonist-

8
P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

Fig. 7. Effect of Dyngo4a treatment on gene expression response to PTH(1–34) and PTHrP(1–141) after wash-out in UMR106-01 cells. A: Experimental design:
UMR106-01 cells were exposed to PTH(1–34) or PTHrP(1–141) either continuously, or for 15 min followed by wash-out. One group of cells treated for 15 min were
also pre-treated with Dyngo4a. Gene responses were assessed at 1 h (Nr4a2 and Il6) and 8 h (Tnfsf11 and Bglap1) after exposure to PTH(1–34) or PTHrP(1–141). B-E:
Responses of Nr4a2 and Il6 at 1 h after treatment and Tnfsf11 and Bglap1 after 8 h of treatment; n = 3, data is mean + SEM. *, p < 0.05; **, p < 0.01; ***,
p < 0.001 vs untreated control under the same conditions by one-way ANOVA.

specific selective activation of PKA isoenzymes can occur [6,84], and
AKAPs (A Kinase Anchoring Proteins) tether relevant signaling com-
ponents to specific subcellular organelles allowing for efficient biolo-
gical responses [85–87]. The main motivation for our study came from
our observation that full length PTHrP is produced by osteocytes, and is
likely to be the paracrine form that targets bone cells [19]. The findings
here suggest that active domains within the carboxy-terminus of the full
molecule could influence interaction with PTH1R in ways that cannot
be achieved with the amino-terminal domain peptides of PTH and
PTHrP. That possibility needs to be investigated.
Because it seemed possible that the effects of PTHrP(1–141) might
result from persistent endosomal signaling, we used a pharmacological
approach to block endocytosis. To exclude contributions of off-target
effects, we used both Pitstop2 and Dyngo4a, and observed similar ef-
fects on cAMP production with both inhibitors including when each
were used at low doses. The endocytosis inhibitor Pitstop2 [73] was
effective in inhibiting the persistent activation response. We also used
Dyngo4a, a small molecule dynamin inhibitor that rapidly and re-
versibly blocks endocytosis [72]. Dynamin is a GTPase that severs
membrane-bound clathrin-coated vesicles. Dyngo4a was developed as
an analog of Dynasore, an earlier dynamin inhibitor that was validated
in a number of cell systems, including inhibition of the TSH receptor
internalization that resulted in persistent activation of adenylyl cyclase
[43]. Treatment with Dyngo4a before wash-out virtually completely
abolished the persistent activation of adenylyl cyclase by PTHrP
(1–141), consistent with the view that the activation resulted from
endocytic signaling. The more profound inhibition by Dyngo4a com-
pared to Pitstop could suggest additional involvement of clathrin-in-
dependent but dynamin-dependent endocytotic mechanism [88] in the
persistent activation response. Dyngo4a treatment of cells also sig-
nificantly prevented the subsequent stimulation of expression of several
genes, particularly genes with a slower response such as Tnfsf11 and
Bglap1, in response to PTHrP(1–141).
We chose to examine the expression of genes known to be influ-
enced by actions through the PTH1R. In UMR106 cells PTHrP rapidly
brought about signaling effects that resulted in later effects on gene
expression that do not require continued presence of agonist. This was
seen also in calvarial osteoblasts. Persistent endosomal signaling
leading to persistent changes in gene expression beyond cAMP stimu-
lation has not been reported previously for the PTH1R but has been
demonstrated in the case of some GPCR agonists. It is necessary for
modulation of cardiac neuron excitability by PACAP [77], of pain
transmission by CGRP [80], and for transcriptional signaling by β-
adrenergic agonists [75].
With increasing evidence that PTHrP is a paracrine and/or auto-
crine regulator of bone remodeling [18] and of the growth and invasive
properties of osteogenic sarcoma [45,52], the question of the nature of
locally generated PTHrP assumes importance. Directly relevant to this
is our recent finding in the immortalized osteocyte cell line, Ocy454,
that the form of PTHrP secreted by osteocytes is full length PTHrP, with
no evidence of secretion of lower molecular weight forms that contain
the N-terminal domain required for action upon PTH1R [19]. That

9
P.W.M. Ho, et al. Biochemical Pharmacology 169 (2019) 113627

A cAMP
formation B * **
300
1 PTH or PTHrP Maximal Response (Max.)

cAMP (pmol/well)
-12 0
minutes

cAMP
200
wash-out formation

2 PTH or PTHrP Response after wash

100
-12 0
minutes
cAMP
wash-out formation

2nd treatment 0
3 PTH or PTHrP PTH or PTHrP Max. After PTHrP PTH Max. After PTHrP PTH
-12 0 -12 16 wash 2nd 2nd wash 2nd 2nd
minutes minutes hours
PTH(1-34) PTHrP(1-141)
cAMP 1st treatment 1st treatment
formation

C wash-out 2nd
treatment
120
PTHrP(1-141)
PTH or PTHrP
or PGE2 D
-12 0 1 cAMP
minutes hour formation 100
wash-out 2nd treatment
PTH or PTHrP
PTHrP(1-141) or PGE2 80 PTH(1-34) 10nM
-12 0 4
% Max. cAMP
cAMP
minutes
wash-out
hours formation * PTHrP(1-141) 10nM
2nd treatment
60 PGE2 100nM
PTH or PTHrP ***
PTHrP(1-141) or PGE2
cAMP
Max. cAMP
-12
minutes
0

wash-out
8
hours
formation
40 ***
2nd treatment

PTHrP(1-141)
PTH or PTHrP
or PGE2
*** ***
20
-12
minutes
0 16
hours
cAMP
formation
***
wash-out 2nd treatment
0
PTH or PTHrP 01 4 8 16 24
PTHrP(1-141) or PGE2
-12 0 24 Time (hr) after wash
minutes hours

Fig. 8. Long-term desensitization of the PTH1R-mediated cAMP formation by PTHrP(1–141). A: Experimental design. UMR106-01 cells were treated with PTH(1–34)
or PTHrP(1–141) (both at 10 nM) for 12 min. Following this cAMP formation was assayed at 3 times: (1) immediately after the treatment to determine maximal
response, (2) 16 h after wash-out of peptide, and (3) following incubation in ligand-free media for 16 h and a second 12 min treatment with 10 nM PTH(1–34) or (4)
PTHrP(1–141). B: cAMP response data for the experiment described in panel A. C,D: Time-course study experimental design. UMR106-01 cells were exposed to
PTHrP(1–141) for 12 min (10 nM), and following wash-out, cells were subjected to a second treatment of either PTHrP(1–141) at 10 nM, PTH(1–34) at 10 nM, or
prostaglandin E2 (PGE2) at 100 nM) at intervals of 1, 4, 8, 16, and 24 h after the initial PTHrP(1–141) exposure. cAMP formation was assayed after this second
exposure. Maximal cAMP response (without PTHrP(1–141) pretreatment was assessed at the same time points to allow correction for diminution of the response
during the time in culture and were as follows: PTHrP(1–141): 190 ± 19; PTH(1–34): 158 ± 16; PGE2: 260 ± 23 pmol/well. Data is mean ± SEM. **, p < 0.01;
***, p < 0.001 vs wash-out without Dyngo by one-way ANOVA.

provided the first study of the nature of PTHrP secreted by cells that use biologically active domains within PTHrP [24,92], full length PTHrP
the constitutive secretory pathway (e.g. cells of mesenchymal origin) has sustained signaling action within PTH1R-expressing cells. Paracrine
rather than those using the regulated secretory pathway that is pro- PTHrP could generate responses that differ from those of the hormone
vided by neuroendocrine cells [89]. Previous studies, using neu- PTH, or of teriparatide (PTH(1–34)) and abaloparatide used in therapy.
roendocrine cells that possess the machinery to package and process M-PTH(1–34) is so very different structurally from PTHrP(1–141) that
proteins in the regulated secretory pathway, suggested that PTHrP the similar action of the two in these experiments has no ready ex-
could be post-translationally processed within the cell to several pep- planation unless it relates directly to the nature of interaction of M-PTH
tides via the regulated secretory pathway [89]. It has been long un- (1–34) with PTH1R [48]. The effect of dynamin blockade on the re-
derstood however, that most cells, particularly those of mesenchymal sponse to M-PTH(1–34) has not been tested, nor has the interaction of
origin use the constitutive secretory pathway, in which no such pro- PTHrP(1–141) with the R0 conformation of PTH1R.
cessing occurs [90]. Thus, the findings in osteocytes make it likely that Properties of PTHrP that are favorable for a paracrine role include
in osteoblast lineage and other cells of mesenchymal origin, including the short half-life of its mRNA and its marked susceptibility to pro-
chondrocytes, the locally secreted PTHrP is of full length. If that is so, teolytic degradation that could ensure its rapid inactivation locally after
the striking developmental phenotype of abnormal cartilage develop- generation and action [93]. The latter property could ensure a short
ment that develops with Pthlh ablation [91] might reflect loss of sig- period of stimulation of cells by the local product, with the effect that
naling from full length PTHrP. PTHrP generated locally requires only brief interaction to initiate ac-
These in vitro studies can be helpful in considering in vivo events, tivity in the cAMP/PKA pathway that can ensure later events progress
where PTHrP acts as a locally generated, paracrine/autocrine effector in nearby (paracrine) or the same (autocrine) PTH1R-positive cells. The
in cartilage and bone, where the likely local ligand is PTHrP of full finding that brief PTHrP treatment can induce a later prolonged de-
length gaining access to target cells. Our data indicates that, in addition sensitization to subsequent activation through PTH1R could also favor
to biological actions of PTHrP mediated by the several other the paracrine role. PTH and shorter PTHrP peptides used

10
P.W.M. Ho, et al.
pharmacologically by intermittent injection can mimic these conditions
and reproduce the PTHrP physiological action. There are no ready ways
to address such questions in vivo in the case of a cytokine such as PTHrP
however. Pharmacological administration of PTHrP would not be
helpful in view of its great susceptibility to proteolysis.
We conclude that full length PTHrP differs from PTH in the manner
in which it promotes formation of cAMP through the PTH1R. Although
both ligands rapidly activate cAMP signalling, PTHrP elicits a sustained
cAMP response, which is still detected for at least 60 min after ex-
posure. This action is dependent on endocytosis, and results in sus-
tained gene responses in the target cells. This difference in action be-
tween the two ligands of the same receptor, provides a potential
mechanism by which brief exposure to locally-derived full length
PTHrP, despite its susceptibility to degradation, can elicit long-lasting
action in target osteoblasts.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the National Health and Medical
Research Council (Australia) through project grant funding to N.A.S.
and T.J.M. and to N.J.P. N.A.S. was supported by an NHMRC Senior
Research Fellowship. A.S.C. was supported by the Christine and T.J.
Martin Fellowship of the Australian and New Zealand Bone and Mineral
Society. St. Vincent’s Institute is supported by the Victorian State
Government Operational Infrastructure Support scheme.
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