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In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-α in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4–6 h of exposure to TNF-α and CHX, and application of FSS (12 dyne/cm²) significantly attenuated this TNF-α-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-α-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-α-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-α. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-α-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway. © 2002 Wiley-Liss, Inc.

Three desmoglein (Dsg) isoforms are expressed in a differentiation-specific fashion in the epidermis, with Dsg2 being basal, Dsg3 (pemphigus vulgaris antigen) basal and spinous, and Dsg1 (pemphigus foliaceus antigen) predominately granular. To better understand the mechanism(s) regulating Dsg isoform expression, we examined the expression pattern of Dsg1, Dsg2, and Dsg3 in normal human epidermal keratinocytes (NHEKs), the immortalized, nontumorigenic HaCaT cell line, and several squamous cell carcinoma cell lines (SCC-9, SCC-12F, SCC-13, and SCC-25). In all cells, the accumulation of high Dsg protein levels required calcium and was not observed in low calcium (0.05–0.07 mM) media. NHEKs expressed Dsg1 in all media tested, consistent with their normal differentiation capacity. HaCaT and SCC-25 also expressed Dsg1; however, the presence of serum in the media dramatically decreased Dsg1 protein levels. Serum also inhibited Dsg1 mRNA levels in HaCaT cells. Dsg1 was not detected in extracts from SCC-9, SCC-12F, and SCC-13 under any conditions. Since activation of protein kinase C (PKC) is involved in keratinocyte differentiation, we evaluated the effects of PKC down-regulation on Dsg isoform expression. Long-term treatment with either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or bryostatin 1 inhibited levels of Dsg1 and Dsg3, but not Dsg2 in NHEKs and HaCaT cells. Chronic TPA also decreased Dsg1 and Dsg3 mRNA levels in NHEKs, further supporting a role for PKC activation in the expression of the suprabasal Dsg1 and Dsg3. These results identify several regulatory mechanisms by which the differentiation-specific pattern of desmosomal cadherins is established in the epidermis.

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5-to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing β-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

Ovarian folliculogenesis is regulated by many endocrine, paracrine and autocrine factors, and as follicular development progresses, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Angiogenesis is tightly regulated by a variety of pro- and anti-angiogenic factors, including vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aberrant angiogenesis occurs in a number of pathological conditions, such as polycystic ovary syndrome (PCOS) and endometriosis, and novel ways of targeting this could lead to new therapeutics. TSP-1 is an anti-angiogenic factor in many physiological systems and has been shown to have anti-angiogenic effects on follicles in vitro. ABT-428898 is an octpeptide mimetic of TSP-1 and we investigated the effect of ABT-898 on follicular angiogenesis using an in vitro angiogenesis assay, prior to studying its effects in vivo in the common marmoset (Callithrix jacchus). Intact preantral/early antral follicles from 21-day-old rat ovaries were cultured for six days in the presence of ABT-898 (0, 0.1, 1, 10, 100 and 1000nM). At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. In a subsequent experiment, the potential of this peptide to inhibit follicular angiogenesis was investigated in vivo, in marmoset monkeys. The marmosets (n=5) were treated with 2.5mg/kg ABT-898 twice daily throughout the follicular phase of the cycle and control marmosets (n=5) were treated with vehicle. On d10 of the follicular phase, which corresponds to the peri-ovulatory period in controls, all animals were injected with 20mg bromodeoxyuridine (BrdU) one hour before removal of ovaries which were fixed, sectioned and stained for haematoxylin and eosin, CD31, BrdU, CD31/BrdU and caspase-3. Treatment with ABT-898 resulted in the suppression of follicular angiogenesis, a reduction in endothelial cell proliferation at the pre- and early antral stages of follicular development and the suppression of granulosa cell proliferation at early preantral, preantral and early antral stages of follicular development. In addition to the inhibition of angiogenesis, ABT-898 treatment also resulted in a significant increase in the number of large preantral and early antral follicles undergoing apoptosis. However, follicle selection and ovulation were not inhibited by treatment, suggesting that the effects of ABT-898 are stage-specific. Taken together, these results indicate that the TSP peptide is capable of having a dual effect by inhibiting follicular angiogenesis and promoting atresia of antral follicles in vivo, but does not prevent ovulation, as has been observed with direct VEGF inhibitors. These results suggest that the TSP peptide could be a novel therapeutic to inhibit abnormal angiogenesis and induce atresia of accumulated follicles in polycystic ovary syndrome. This research was supported by a Medical Research Council Ph.D. studentship and a core grant to Medical Research Council Reproductive Sciences Unit (U.1276.00.002.00006.01).(poster)