Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36)

Protein kinase C (PKC) signaling can be activated rapidly by 17β-estradiol (E(2)) via nontraditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERβ. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [(3)H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.     

Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.     

Beta-1 integrins mediate substrate dependent effects of 1alpha,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. beta(1) integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1alpha,25(OH)(2)D(3) in a surface-dependent manner. To determine if beta(1) has a role in mediating osteoblast response, we silenced beta(1) expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human beta(1) to block ligand binding. beta(1)-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta(1), prostaglandin E(2), and osteoprotegerin in comparison with control cells. Moreover, beta(1)-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)(2)D(3). Anti beta(1) antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1alpha,25(OH)(2)D(3) on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta(1) plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)(2)D(3). The results also show that beta(1) mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)(2)D(3).     

Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)₂D₃ initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3 ^+/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3 ^+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3 ^+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3 ^+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3 ^+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)₂D₃’s actions in regulating skeletal development.     

BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner

Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast‐like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG‐MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z‐7‐oxozeaenol, dorsomorphin, H‐8). Apoptosis was characterized by caspase‐3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell‐type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG‐MG63 than MG63 cells. 5Z‐7‐oxozeaenol and dorsomorphin eliminated the BMP2‐induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications. J. Cell. Biochem. 113: 3236–3245, 2012. © 2012 Wiley Periodicals, Inc.     

Sex dependent regulation of osteoblast response to implant surface properties by systemic hormones

Background

Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)₂D₃ and 17β-oestradiol (E₂)] and microstructured surfaces on osteoblasts are sex dependent.

Methods

Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)₂D₃, E₂, or E₂ conjugated to bovine serum albumin (E₂-BSA).

Results

Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-β1, osteoprotegerin and prostaglandin E₂ in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E₂ and E₂-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)₂D₃ increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells.

Conclusions

Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)₂D₃ had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones.     

Human osteoblasts exhibit sexual dimorphism in their response to estrogen on microstructured titanium surfaces

Disruption of axonal transport plays a pivotal role in diabetic neuropathy. A sex-dimorphism exists in the incidence and symptomatology of diabetic neuropathy; however, no studies so far have addressed sex differences in axonal motor proteins expression in early diabetes as well as the possible involvement of neuroactive steroids. Interestingly, recent data point to a role for mitochondria in the sexual dimorphism of neurodegenerative diseases. Mitochondria have a fundamental role in axonal transport by producing the motors’ energy source, ATP. Moreover, neuroactive steroids can also regulate mitochondrial function. Here, we investigated the impact of short-term diabetes in the peripheral nervous system of male and female rats on key motor proteins important for axonal transport, mitochondrial function, and neuroactive steroids levels. We show that short-term diabetes alters mRNA levels and axoplasm protein contents of kinesin family member KIF1A, KIF5B, KIF5A and Myosin Va in male but not in female rats. Similarly, the expression of peroxisome proliferator-activated receptor γ co-activator-1α, a subunit of the respiratory chain complex IV, ATP levels and the key regulators of mitochondrial dynamics were affected in males but not in females. Concomitant analysis of neuroactive steroid levels in sciatic nerve showed an alteration of testosterone, dihydrotestosterone, and allopregnanolone in diabetic males, whereas no changes were observed in female rats. These findings suggest that sex-specific decrease in neuroactive steroid levels in male diabetic animals may cause an alteration in their mitochondrial function that in turn might impact in axonal transport, contributing to the sex difference observed in diabetic neuropathy.