Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106-01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter-luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2. 8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106-01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the "parachute assay," which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well-coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106-01 cells. The stretchinduced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady-state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell-to-cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106-01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43-specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43. (J Bone Miner Res 1998; 13:218-228)

Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.

Because lifelong exposure to estrogen is a strong determinant of bone mass, we asked whether metabolic conversion of estrogen to either inactive or active metabolites would reflect postmenopausal bone mineral density (BMD) and rate of bone loss. Biochemical markers of inactive estrogen metabolites, urinary 2-hydroxyestrogen (2OHE 1 ) and 2-methoxyestrogen (2MeOE 1 ), and active metabolites, urinary 16␣-hydroxyestrone (16␣OHE 1 ), estradiol (E 2 ), and estriol (E 3 ), were determined in 71 untreated, healthy postmenopausal women (age, 47-59 years) followed prospectively for 1 year. Urinary 2MeOE 1 was correlated negatively with baseline vertebral (anteroposterior [AP] projection, r ‫؍‬ ؊0.23 and p < 0.05; lateral view, r ‫؍‬ ؊0.27 and p < 0.05) and proximal femur bone density measured by dual-energy X-ray absorptiometry (DXA; total, r ‫؍‬ ؊0.38 and p < 0.01; neck, r ‫؍‬ ؊0.28 and p ‫؍‬ 0.02; trochanter, r ‫؍‬ ؊0.44 and p < 0.01). BMDs of women in the lowest quartile of urinary 2MeOE 1 (<15 ng/g) were significantly higher than those in the highest quartile at all skeletal sites (p < 0.05). Likewise, women in the lowest quartile of urinary 2OHE 1 /16␣OHE 1 ratio (<1.6) did not experience bone loss after 1 year, in contrast to women in the higher quartiles. We propose that the rate of inactivation of estrogens through 2-hydroxylation may contribute to postmenopausal osteoporosis. (J Bone Miner Res 2000;15:2513-2520)

Intermittent administration of parathyroid hormone (PTH) peptides increases bone density in animal and human models of osteoporosis. In vitro studies have demonstrated that PTH analogs lacking the first two amino acids can stimulate cell proliferation in certain cell systems, whereas fragments with an intact N terminus can be antimitogenic. We have tested whether the truncated PTH(3-38) fragment may be a better "anabolic analog" than PTH(1-38) by monitoring bone density and biomechanical properties of the femur in 6-month-old ovariectomized (OVX) rats. Either PTH fragment was administered subcutaneously (8 g/100 g of body weight) 5 days/week, for 4 weeks, starting 1 week after surgery. During the entire study, untreated OVX rats lost 12.1 ؎ 4.4% of their initial bone density. PTH(1-38) reversed the initial bone loss, leading to complete restoration of presurgery values after 4 weeks of treatment. Conversely, administration of PTH(3-38) resulted in 13.2 ؎ 5.8% bone loss, while continuous estrogen infusion (10 g/kg/day) prevented bone loss but did not reverse it. Sham-operated animals also experienced significant bone loss in the vehicle and PTH(3-38)-treated groups (؊4.5 ؎ 6.7%, and ؊7.6 ؎ 2.8%, respectively), whereas a significant gain in bone density (؉4.4 ؎ 5.6%) was observed in the rats treated with PTH(1-38). A bone quality factor (index of strain energy loss) and the impact strength (resistance to fracture) were 25% and 44% lower in femurs explanted from OVX animals treated with either vehicle or PTH(3-38), compared with sham-operated animals. On the contrary, no difference was observed between OVX and control animals after treatment with PTH(1-38), indicating a preservation of the capacity to withstand mechanical stress. Thus, PTH(1-38) counteracts estrogen-dependent loss of mineral density and bone biomechanical properties and increases bone density in estrogen-replete animals. An intact N terminus sequence is necessary for this anabolic action of PTH. (J Bone Miner Res 1997;12:384-392)

As human beings venture into space in the 21st century, they will be confronted with a "hypodynamic" and thus hostile environment for the bone homeostasis, that could potentially compromise their mobility in general and skeletal strength in particular after landing. From this point of view, space flight studies are especially interesting and intriguing models for scientists. Space studies, however, must not only overcome enormous technical problems but are also limited in size and frequency. Therefore, ground-based models have also been developed to evaluate the effects of skeletal unloading. The most popular model for human studies is prolonged bed rest with normal volunteers, although studies with paraplegics have also been undertaken. In animals, the hindlimb elevation (tail suspension) model simulates space flight models and is well tolerated by the animals with minimal evidence of stress. Although negative calcium balance and bone loss have been observed in all the aforementioned models of skeletal unloading, the exact mechanism(s) by which this occurs are still unknown and mainly speculative.

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