In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity

Murrills, Richard J.; Matteo, Jeanne J.; Samuel, Rachelle L.; Andrews, Jennifer L.; Bhat, Bheem M.; Coleburn, Valerie E.; Kharode, Yogendra; Bex, Frederick J.

doi:10.1016/j.bone.2004.08.015

Cited by 12 publications

(17 citation statements)

References 39 publications

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“…Bioequivalent doses of the two analogs were selected on the basis of comparable stimulation of plasma cAMP levels 10 min after sc peptide administration and of cAMP-dependent mRNA upregulation in long bones at a later time (6 h). Further, the acute cAMP responses to PTH(1-34) and each of the two G 1 ,R 19 -substituted analogs at the conclusion of the 4-week anabolic protocol were found to have remained comparable and, in contrast to the loss of cAMP responsiveness in isolated osteoblasts induced by continuous PTH exposure in vitro [12,23], were not desensitized relative to the response seen in control animals not previously exposed to exogenous PTH. We have strived to carefully establish in vivo bioequivalent doses for the three PTH peptides used, with respect to the cAMP response that they share in common.…”

Section: Discussionmentioning

confidence: 89%

“…In this regard, PKC signaling via PTH1Rs has been related to induction of osteoblastic bone formation [16,24], especially PKC stimulated by the PTH(29-34) portion of the ligand [14,21]. G 1 ,R 19 (1-28) lacks the 29-34 domain of hPTH(1-34) required for PLC-independent PKC activation and incorporates a Ser 1 to Gly 1 mutation that blocks PLC activation (the Arg 19 change improves binding affinity that otherwise is compromised by the C-terminal truncation) [27]. This peptide can no longer activate PKC via either PLC-dependent or PLC-independent mechanisms but can fully activate cAMP production via PTH1Rs, albeit with somewhat reduced potency [26,31].…”

Section: Discussionmentioning

confidence: 99%

“…This peptide can no longer activate PKC via either PLC-dependent or PLC-independent mechanisms but can fully activate cAMP production via PTH1Rs, albeit with somewhat reduced potency [26,31]. The related G 1 ,R 19 (1-34) analog, which retains the 29-34 domain, was shown to activate both cAMP/ PKA and (PLC-independent) PKC but is incapable of PLC activation [31]. The strategy used in the current study was to administer each of these analogs to mice, in an intermittent-treatment protocol, at doses that would activate cAMP/PKA signaling in vivo to the same extent as standard PTH(1-34) (40 μg/kg/d) and then to seek differences in skeletal responses that could be attributable to loss of one or both of the PKC-activation mechanisms normally available to the PTH1R in bone cells.…”

Section: Discussionmentioning

confidence: 99%

“…Further, carboxyl-terminal truncation, to remove the sequence His 32 -Asn 33 -Phe 34 (i.e., hPTH ), precludes activation of membrane-associated PKC(s) but does not impair stimulation of adenylyl cyclase or eliminate the anabolic response in vivo, which has further supported the role of cAMP in the anabolic response [13,22,26,27]. Other evidence suggests that PTH1R-dependent adenylyl cyclase activation alone may not be sufficient to elicit a full anabolic response, however [18,19], and interpretation of the studies with amino-truncated analogs was clouded by the lack of recognition that the PLC/PKC response to PTH is critically dependent upon the presence of the α-amino group of the amino-terminal Ser 1 of hPTH(1-34) -i.e., inactive amino-truncated fragments actually lack both adenylyl cyclase and PLC/PKC responses [26]. Thus, the question of the relative roles of the cAMP vs. PKC-activating PTH1R signaling pathways in mediating the anabolic action of the hormone in vivo remains unsettled.…”

Section: Introductionmentioning

confidence: 99%

“…In the present experiments, we employed two signaling-selective peptides, [Gly 1 , Arg 19 ]hPTH and [Gly 1 ,Arg 19 ]hPTH(1-34), together with PTH(1-34), to investigate, in vivo, the anabolic effects of different signaling pathways generated by PTH and to test the hypothesis that PTH1R-generated PKC signaling, arising via either PLC-dependent or PLC-independent mechanisms, contributes to the anabolic response of bone.…”

Section: Introductionmentioning

confidence: 99%

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Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone

Yang

Singh

Divieti

et al. 2007

Bone

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PTH regulates osteoblastic function by activating PTH/PTHrP receptors (PTH1Rs), which trigger several signaling pathways in parallel, including cAMP/protein kinase A (PKA) and, via both phospholipase-C (PLC)-dependent and PLC-independent mechanisms, protein kinase C (PKC). These signaling functions have been mapped to distinct domains within PTH(1-34), but their roles in mediating the anabolic effect of intermittent PTH in vivo are unclear. We compared the anabolic effects in mice of hPTH(1-34) with those of two analogs having restricted patterns of PTH1R signaling. [G 1 ,R 19 ]hPTH(1-28) lacks the 29-34 domain of hPTH(1-34) needed for PLCindependent PKC activation, incorporates a Gly 1 mutation that prevents PLC activation, and stimulates only cAMP/PKA signaling. [G 1 ,R 19 ]hPTH(1-34) retains the 29-34 domain and activates both cAMP/PKA and PLC-independent PKC.Human PTH(1-34) (40 μg/kg), [G 1 ,R 19 ]hPTH(1-34) (120 μg/kg), and [G 1 ,R 19 ]hPTH(1-28) (800 μg/kg), at doses equipotent in elevating blood cAMP at 10 min and cAMP-dependent gene expression in bone at 6 h after s.c. injection, were administered to 10 week old female C57BL/6J mice 5 days/ week for 4 weeks. Acute blood cAMP responses, retested after 4 weeks, were not reduced by the preceding PTH treatment. The three PTH peptides induced equivalent increases in distal femoral bone mineral density (BMD), and, by microCT analysis, distal femoral and vertebral bone volume and trabecular thickness and mid-femoral cortical endosteal apposition. [G 1 ,R 19 ]hPTH(1-34) and hPTH(1-34) increased distal femoral BMD more rapidly and augmented total-body BMD and bone volume of proximal tibial trabeculi to a greater extent than did [G 1 ,R 19 ]hPTH(1-28),.We conclude that cAMP/PKA signaling is the dominant mechanism for the anabolic actions of PTH in trabecular bone and PLC-independent PKC signaling, attributable to the PTH(29-34) sequence, appears to accelerate the trabecular response and augment BMD at some skeletal sites. PTH1R PLC signaling pathway is not required for an anabolic effect of intermittent PTH(1-34) on bone.

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone

Yang

Singh

Divieti

et al. 2007

Bone

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Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Murrills¹,

Andrews²,

Samuel³

et al. 2009

J of Cellular Biochemistry

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Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE-Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos-2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH-induced activation of the CRE. Activators of non-cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE-luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H-89 (10 microM) almost completely blocked PTH-induced activation of the CRE-reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time-dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH-induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN-93 had no significant effect on the response to PTH. We conclude that non-cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH.

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Utility of the Ovariectomized Rat as a Model for Human Osteoporosis in Drug Discovery

Kharode¹,

Sharp²,

Bodine³

2008

Osteoporosis

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Ovariectomy-induced osteopenia in the rat produces skeletal responses similar to that in a post-menopausal woman. In the ovariectomized (ovx) rat, high bone turnover, and subsequent bone loss, like in the human post-menopausal condition, can be prevented by estrogen replacement. Because of the striking resemblance of skeletal responses in humans and rats in the state of estrogen deficiency, the ovx rat is considered to be a gold standard model for evaluating drugs for prevention and reversal of osteoporosis. This chapter describes the procedure for performing ovariectomy on the rat and the utility of the ovx rat model we have utilized over the last two decades in our laboratory.

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In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity

Cited by 12 publications

References 39 publications

Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone

Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Utility of the Ovariectomized Rat as a Model for Human Osteoporosis in Drug Discovery

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