S. MacLean

Comparison of the ability of recombinant human parathyroid hormone, rhPTH-(1-84), and hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats

A recombinant human parathyroid hormone, rhPTH-(1–84), which is currently in Phase II clinical trial, and hPTH-(1–31)NH2 (Ostabolin) are promising anabolic agents for treating osteoporosis because they can stimulate cortical and trabecular bone growth in osteopenic, ovariec-tomized (OVX) rats and in osteoporotic, postmenopausal women when injected subcutaneously and intermittently at low doses. We have now found that, despite their different sizes and signaling properties (rhPTH-(1–84) stimulates adenylyl cyclase and phospholipase C; hPTH-(1–31)NH2 only stimulates adenylyl cyclase), they are equally osteogenic in OVX rats. Thus daily subcutaneous injections of 0.6 nmol/ 100 g of body weight of rhPTH-(1–84) or hPTH-(1–31)NH2 into 3-month-old OVX rats for 6 weeks starting 2 weeks after OVX equally reduced the otherwise large OVX-triggered loss of femoral trabecular bone. Daily subcutaneous injections of 0.4 or 0.8 nmol/100 g of body weight of the two agents for 6 weeks also equally increased the mean thickness of the remaining femoral trabeculae in 3-month-old and 1-year-old OVX rats to 20 to 80% above the value in normal animals when started 9 weeks after ovariectomy.

Cyclization by a specific lactam increases the ability of human parathyroid hormone (hPTH)-(1-31)NH2 to stimulate bone growth in ovariectomized rats

Human parathyroid hormone (hPTH)‐(1–31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase‐C as do hPTH(1–84) and hPTH(1–34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side‐chain lactams have been introduced in the hydrophilic face of the receptor‐binding region of the fragments Ser17‐Val31 amphiphilic α‐helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this α‐helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2, which had six times the AC‐stimulating ability of hPTH(1–31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix‐stabilizing lactam derivative [Leu27]‐cyclo(Lys26‐Arg30)‐hPTH(1–31)NH2 did not have a greater AC‐stimulating ability than hPTH(1–31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTHs anabolic action, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1–31)NH2 or [Leu27]‐cyclo(Lys26‐Arg30)‐hPTH(1–31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1–31)NH2, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1–34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase Cs (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH‐related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)‐terminally truncated fragments of PTH(1–34), such as human PTH(1–31) [hPTH‐(1–31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1–31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)‐terminally truncated fragments, such as bovine PTH(3–34) [bPTH(3–34)], hPTH(3–34)NH2, and hPTH(13–34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N‐terminally truncated analogs bPTH(3–34)NH2 and hPTH(13–34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1–34)NH2 and hPTH(1–31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N‐terminally truncated fragments stimulate PKC activity as strongly as hPTH(1–34)NH2 and hPTH(1–31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C‐terminally truncated fragments of PTH(1–34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1‐transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N‐terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.

Intrinsic Role of FoxO3a in the Development of CD8 + T Cell Memory

CD8+ T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8+ T cells. The mechanisms that govern the massive contraction and maintenance of primed CD8+ T cells are not clear. We show in this study that the transcription factor, FoxO3a, does not influence Ag presentation and the consequent expansion of CD8+ T cell response during Listeria monocytogenes infection, but plays a key role in the maintenance of memory CD8+ T cells. The effector function of primed CD8+ T cells as revealed by cytokine secretion and CD107a degranulation was not influenced by inactivation of FoxO3a. Interestingly, FoxO3a-deficient CD8+ T cells displayed reduced expression of proapoptotic molecules BIM and PUMA during the various phases of response, and underwent reduced apoptosis in comparison with wild-type cells. A higher number of memory precursor effector cells and memory subsets was detectable in FoxO3a-deficient mice compared with wild-type mice. Furthermore, FoxO3a-deficient memory CD8+ T cells upon transfer into normal or RAG1-deficient mice displayed enhanced survival. These results suggest that FoxO3a acts in a cell-intrinsic manner to regulate the survival of primed CD8+ T cells.

CD8+ T Cells Primed in the Periphery Provide Time-Bound Immune-Surveillance to the Central Nervous System

After vaccination, memory CD8+ T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8+ T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8+ T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8+ T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8+ T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8+ T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8+ T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69hi) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69low and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8+ T cells that do not reside in the parenchyma.

The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats

The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding α-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH’s anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam linkages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.

Lactam Formation Increases Receptor Binding, Adenylyl Cyclase Stimulation and Bone Growth Stimulation by Human Parathyroid Hormone (hPTH)(1–28)NH2

Human parathyroid hormone (1–28)NH2 [hPTH(1–28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast‐like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I‐[Nle8,18, Tyr34]bovine PTH(1–34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH‐related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the α‐helix in the residue 17–28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22‐Lys26)hPTH(1–28)NH2 analog dramatically reduced the IC50 for displacing 125I‐[Nle8,18, Tyr34]bPTH(1–34)NH2 from hPTHIRs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1–28)NH2. Thus, hPTH(1–28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22‐Lys26)hPTH(1–28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1–28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs. (J Bone Miner Res 2000;15:964–970)

The Stimulation of Vertebral and Tibial Bone Growth by the Parathyroid Hormone Fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2

Abstract. The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10–25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1–L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.

Stimulation of Femoral Trabecular Bone Growth in Ovariectomized Rats by Human Parathyroid Hormone (hPTH)-(1-30)NH2

Abstract. It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTHs bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH2, which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH2 to stimulate bone growth despite its having hPTH-(1-31)NH2s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH2s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragments ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2–10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10–50 nmol of hPTH-(1-30)NH2/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH2/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, hPTH-(1-34)NH2 and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)NH2. These results have removed a major objection to the proposal that PTHs osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause.

Comparison of the Abilities of Human Parathyroid Hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to Stimulate Femoral Trabecular Bone Growth in Ovariectomized Rats

Abstract. hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.