R. J. Isaacs

Control of 3T3 cell proliferation by calcium.

SummaryWhen a population of 3T3 mouse cells was subcultured regularly at confluency, the original epitheliodid or stellate cells disappeared and, by the ninth passage, they had been replaced by spindle-shaped cells. The original cells proliferated only when the extracellular calcium concentration exceeded 0.1mm, and their proliferative activity became maximum only when the calcium concentration was 0.5mm. The spindle-shaped cells were much more sensitive to proliferative stimulation by calcium. Although these cells also could not proliferate without extracellular ionic calcium, they proliferated maximally in the presence of as little as 0.05mm calcium. Thus, calcium is a major regulator of the proliferation of 3T3 mouse cells. Moreover, it appears that the sensitivity of the proliferative machinery to the calcium ion can vary greatly within an established cell line.

STIMULATION OF THE GROWTH OF FEMORAL TRABECULAR BONE IN OVARIECTOMIZED RATS BY THE NOVEL PARATHYROID HORMONE FRAGMENT, HPTH-(1-31)NH2 (OSTABOLIN)

The human parathyroid hormone, hPTH-(1–84), and its hPTH-(1–34) fragment are promising anabolic agents for treating osteoporosis because they can strongly stimulate the production of biomechanically effective cortical and trabecular bone in osteopenic ovariectomized (OVX) rats and trabecular bone in osteoporotic postmenopausal humans. The ideal PTH fragment for treating osteoporosis would be the smallest and functionally simplest fragment that activates only one signal mechanism and still strongly stimulates trabecular bone growth. A new PTH fragment, hPTH-(1–31)NH2, which only stimulates adenylyl cyclase instead of stimulating both adenylyl cyclase and phospholipase-C as do hPTH-(1–84) and hPTH-(1–34), is this minimum, high-potency anabolic fragment. hPTH-(1–31)NH2 (which we have named Ostabolin) can greatly thicken trabeculae and increase the dry weight and calcium content of trabecular bone in the distal femurs of osteopenic, young, sexually mature OVX Sprague-Dawley rats when injected subcutaneously each day for 6 weeks at doses between 0.4 and 1.6 nmole/100 g of body weight.

The different roles of serum and calcium in the control of proliferation of balb/c 3t3 mouse cells.

Proliferatively inactive BALB/c 3T3 mouse cells in dense cultures initiate a growth-division cycle upon exposure to fresh calf serum in a low-calcium (0.01 µM) medium. If these calcium-deprived cells are not supplied with calcium sometime during the first 10 hours after serum stimulation, they will rapidly return to a proliferatively inactive state without initiating DNA synthesis. The prereplicative development of such stimulated calcium-deprived cells appears to stop at an advanced stage, because addition of calcium as late as 10 hours after serum exposure rapidly initiates DNA synthesis, and enables the culture’s DNA-synthetic activity subsequently to reach its peak value at the same time as in control cultures.

Stimulation of Protein Kinase C during Ca-induced Keratinocyte Differentiation SELECTIVE BLOCKADE OF MARCKS PHOSPHORYLATION BY CALMODULIN

Raising the external Ca concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca- or TPA-stimulated keratinocytes. Raising the external Ca concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. LopezBarahona, M., and Aaronson, S. A.(1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca did not. Furthermore, Ca-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca•CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca. Ca•calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca•calmodulin complexes, formed by the internal Ca surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.

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.

C-terminal fragment of parathyroid hormone-related protein, PTHrP-(107-111), stimulates membrane-associated protein kinase C activity and modulates the proliferation of human and murine skin keratinocytes.

Low concentrations of the C‐terminal parathyroid hormone‐related protein (PTHrP) fragments, PTHrP‐(107–111) and PTHrP‐(107–139), stimulated membrane‐associated protein kinase Cs (PKCs), but not adenylyl cyclase or an internal Ca2+ surge, in early passage human skin keratinocytes and BALB/MK‐2 murine skin keratinocytes. The fragment maximally stimulated membrane‐associated PKCs in BALB/MK‐2 cells at 5 × 10−9 to 10−8 M. The maximally PKC‐stimulating concentrations of PTHrP‐(107–111) also stopped or stimulated BALB/MK‐2 keratinocyte proliferation depending on whether the cells were, respectively, cycling or quiescent at the time of exposure. Thus, just one brief (30‐minute) pulse of 10 −8 M PTHrP‐(107–111) stopped the proliferation of BALB/MK‐2 keratinocytes for at least 5 days. On the other hand, daily 30‐minute pulses of 10−8 M PTHrP‐(107–111) started and then maintained the proliferation of initially quiescent BALB/MK‐2 cells. Similarly PTHrP‐(107–111) inhibited DNA synthesis by cycling primary adult human keratinocytes, but it stimulated DNA synthesis by quiescent human keratinocytes.

Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone-(1–34)

It is commonly believed that the parathyroid hormones (PTHs) main function in bone is to stimulate osteoclastic resorption. However, intermittent injections of small doses of PTH holoprotein, but more often its bioactive hPTH-(1–34) fragment, have been shown to stimulate bone growth in animals and humans through their ability to stimulate adenylyl cyclase and not their ability to independently activate a protein kinases-C stimulating mechanism. This anabolic action suggests that PTH might be an effective therapeutic for osteoporosis. If so, the hormone must be able to restore severely depleted trabecular bone, and the goal of this study was to find out if it can. To do this, we started a multiweek program of daily subcutaneous injections of 0.8 nmoles of hPTH-(1–34)/100 g body weight into rats at 4, 8, or 16 weeks after ovariectomy (OVX) and the increasingly severe selective loss of trabecular bone. These injections strongly stimulated femoral trabecular bone to grow and mineralize at the same rate regardless of how much of it had been lost before the injections were started. Thus, the progressively depleting trabecular bone in the femurs of OVX rats does not lose its anabolic responsiveness to PTH. This finding is another indication of the likelihood of small, adenylyl cyclase-stimulating PTH fragments being effective therapeutics for osteoporosis.

An examination of the roles of cyclic nucleotides in the initiation of cell proliferation

Abstract The initiation of proliferative activity in proliferatively quiescent confluent monolayers of BALB/3T3 cell cultures by a fresh medium-serum replacement is independent of early (within minutes) fluctuations in total cellular cyclic AMP or cyclic GMP levels. However, the possibility of a later prereplicative cyclic AMP surge being involved in the initiation of DNA synthesis will be discussed.

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.

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.