Dopamine Alleviates Phloridzin Toxicity in Apple by Modifying Rhizosphere Bacterial Community Structure and Function.

Phloridzin significantly influences apple plant growth, development, and resistance to environmental stresses by engaging in various metabolic processes. Its excessive accumulation in soil, attributed to continuous monoculture practices, not only inhibits plant growth but also disrupts the rhizosphere microbial community. This study aims to explore the remedial effects of dopamine, a known antioxidant and stress resistance modulator in plants, on the adverse impacts of phloridzin stress in apple. Through hydroponic and pot experiments, it was demonstrated that dopamine significantly mitigates the growth inhibition caused by phloridzin stress in apple by reducing reactive oxygen species levels and enhancing photosynthesis and nitrogen transport. Additionally, dopamine reduced phloridzin concentrations in both the rhizosphere and roots. Furthermore, dopamine positively influences the structure of the rhizosphere microbial community, enriching beneficial microbes associated with nitrogen cycling. It increases the potential for soil nitrogen degradation and fixation by upregulating the abundance of ureC, GDH, and nifH, as revealed by metagenomic analysis. This aids in alleviating phloridzin stress. The study reveals dopamine's pivotal roles in modulating rhizosphere ecology under phloridzin stress and suggests its potential in sustainable apple cultivation practices to counter ARD and enhance productivity.

Transcriptome and metabolome analyses reveal the regulatory role of MdPYL9 in drought resistance in apple.

BACKGROUND: The mechanisms by which the apple MdPYL9 gene mediates the response to drought stress remain unclear. Here, transcriptome and metabolome analyses of apple plants under drought were used to investigate the mechanisms by which MdPYL9 regulates the response to drought stress in apple. MdPYL9-overexpressed transgenic and non-transgenic apple histoculture seedlings were rooted, transplanted, and subjected to drought treatments to clarify the mechanisms underlying the responses of apples to drought stress through phenotypic observations, physiological and biochemical index measurements, and transcriptomic and metabolomic analyses. RESULTS: Under drought stress treatment, transgenic plants were less affected by drought stress than non-transgenic plants. Decreases in the net photosynthetic rate, stomatal conductance, and transpiration rate of transgenic apple plants were less pronounced in transgenic plants than in non-transgenic plants, and increases in the intercellular CO2 concentration were less pronounced in transgenic plants than in non-transgenic plants. The relative electrical conductivity and content of malondialdehyde, superoxide anion, and hydrogen peroxide were significantly lower in transgenic plants than in non-transgenic plants, and the chlorophyll content and activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were significantly higher in transgenic plants than in non-transgenic plants. The number of differentially expressed genes (DEGs) involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and the most significant and highly annotated DEGs in the transgenic plants were involved in the flavonoid biosynthesis pathway, and the most significant and highly annotated DEGs in control plants were involved in the phytohormone signal transduction pathway. The number of differentially accumulated metabolites involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and up-regulated metabolites were significantly enriched in apigenin-7-O-glucoside in transgenic plants and in abscisic acid in non-transgenic plants. In the flavonoid biosynthetic pathway, the expression of genes encoding chalcone synthase (CHS) and chalcone isomerase (CHI) was more significantly down-regulated in non-transgenic plants than in transgenic plants, and the expression of the gene encoding 4-coumarate-CoA ligase (4CL) was more significantly up-regulated in transgenic plants than in non-transgenic plants, which resulted in the significant up-regulation of apigenin-7-O-glucoside in transgenic plants. CONCLUSIONS: The above results indicated that the over-expression of MdPYL9 increased the drought resistance of plants under drought stress by attenuating the down-regulation of the expression of genes encoding CHS and CHI and enhancing the up-regulated expression of the gene encoding 4CL, which enhanced the content of apigenin-7-O-glucoside.

Melatonin promotes the recovery of apple plants after waterlogging by shaping the structure and function of the rhizosphere microbiome.

The dynamics of the physiological adaptability of plants and the rhizosphere soil environment after waterlogging remain unclear. Here we investigated the mechanisms regulating plant condition and shaping of the rhizosphere microbiome in a pot experiment. In the experiment, we added melatonin to waterlogged plants, which promoted waterlogging relief. The treatment significantly enhanced photosynthesis and the antioxidant capacity of apple plants, and significantly promoted nitrogen (N) utilization efficiency by upregulating genes related to N transport and metabolism. Multiperiod soil microbiome analysis showed the dynamic effects of melatonin on the diversity of the microbial community during waterlogging recovery. Random forest and linear regression analyses were used to screen for potential beneficial bacteria (e.g., Azoarcus, Pseudomonas and Nocardioides) specifically regulated by melatonin and revealed a positive correlation with soil nutrient levels and plant growth. Furthermore, metagenomic analyses revealed the regulatory effects of melatonin on genes involved in N cycling in soil. Melatonin positively contributed to the accumulation of plant dry weight by upregulating the expression of nifD and nifK (N fixation). In summary, melatonin positively regulates physiological functions in plants and the structure and function of the microbial community; it promoted the recovery of apple plants after waterlogging stress.

Integrative physiological, metabolomic, and transcriptomic analysis reveals the drought responses of two apple rootstock cultivars.

BACKGROUND: Drought is considered the main environmental factor restricting apple production and thus the development of the apple industry. Rootstocks play an important role in enhancing the drought tolerance of apple plants. Studies of the physiology have demonstrated that 'ZC9-3' is a strong drought-resistant rootstock, whereas 'Jizhen-2' is a weak drought-resistant rootstock. However, the metabolites in these two apple rootstock varieties that respond to drought stress have not yet been characterized, and the molecular mechanisms underlying their responses to drought stress remain unclear. RESULTS: In this study, the physiological and molecular mechanisms underlying differences in the drought resistance of 'Jizhen-2' (drought-sensitive) and 'ZC9-3' (drought-resistant) apple rootstocks were explored. Under drought stress, the relative water content of the leaves was maintained at higher levels in 'ZC9-3' than in 'Jizhen-2', and the photosynthetic, antioxidant, and osmoregulatory capacities of 'ZC9-3' were stronger than those of 'Jizhen-2'. Metabolome analysis revealed a total of 95 and 156 differentially accumulated metabolites in 'Jizhen-2' and 'ZC9-3' under drought stress, respectively. The up-regulated metabolites in the two cultivars were mainly amino acids and derivatives. Transcriptome analysis revealed that there were more differentially expressed genes and transcription factors in 'ZC9-3' than in 'Jizhen-2' throughout the drought treatment. Metabolomic and transcriptomic analysis revealed that amino acid biosynthesis pathways play key roles in mediating drought resistance in apple rootstocks. A total of 13 metabolites, including L-α-aminoadipate, L-homoserine, L-threonine, L-isoleucine, L-valine, L-leucine, (2S)-2-isopropylmalate, anthranilate, L-tryptophan, L-phenylalanine, L-tyrosine, L-glutamate, and L-proline, play an important role in the difference in drought resistance between 'ZC9-3' and 'Jizhen-2'. In addition, 13 genes encoding O-acetylserine-(thiol)-lyase, S-adenosylmethionine synthetase, ketol-acid isomeroreductase, dihydroxyacid dehydratase, isopropylmalate isomerase, branched-chain aminotransferase, pyruvate kinase, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase, N-acetylglutamate-5-P-reductase, and pyrroline-5-carboxylate synthetase positively regulate the response of 'ZC9-3' to drought stress. CONCLUSIONS: This study enhances our understanding of the response of apple rootstocks to drought stress at the physiological, metabolic, and transcriptional levels and provides key insights that will aid the cultivation of drought-resistant apple rootstock cultivars. Especially, it identifies key metabolites and genes underlying the drought resistance of apple rootstocks.

Effects of different dwarfing interstocks on the rhizosphere, endophytic bacteria, and drought resistance of apple trees.

Rootstock is commonly used to enhance plant resistance to drought stress. However, it is necessary to investigate the effects of different rootstock, interstock, and scion combinations on rhizosphere and root endophytic bacteria under drought stress. We conducted a pot experiment to investigate how interstock [SH40, Jizhen 1 (J1), and Jizhen 2 (J2)] affects the drought tolerance and nitrogen (N) uptake and utilization of apple trees under drought stress. The results showed that the total dry weight, total chlorophyll content, carotenoid content, photosynthesis rate, and N absorption and utilization efficiency of apple trees decreased significantly, whereas relative electrolyte leakage increased significantly under drought stress. Membership function analysis showed that the apple plants with the J1 interstock had the greatest drought resistance. In addition, drought treatment significantly affected the diversity and composition of rhizosphere and root endophytic communities in all three rootstock/interstock/scion combinations. Further analysis revealed that the relative abundance of the plant pathogen Ralstonia was significantly increased in J2 drought-treated roots, compared to the other groups, whereas those of some potentially beneficial bacteria (0134_terrestrial_group, Phenylobacterium, Ellin6067, Kribbella, Chloronema, and Streptomyces) increased significantly in the J1 drought-treated sample. Co-occurrence network analysis showed that some potentially beneficial bacteria (Ellin6067, S0134_terrestrial_group, Pedomicrobium, and Subgroup_10) were significantly positively correlated with N content. These modifications of the rhizosphere and endophytic bacterial communities may influence the drought resilience and N uptake efficiency of different combinations of interstocks and scions. This study is a much-needed step towards understanding the stress response mechanism of scion-rootstock combinations.

Exogenous melatonin alleviates apple replant disease by regulating rhizosphere soil microbial community structure and nitrogen metabolism.

Apple replant disease (ARD) is a common soil-borne disease afflicting apple plants. Melatonin is a broad-spectrum oxygen scavenger that plays a key role in alleviating stress-induced damage in plants. In this study, we aimed to determine whether adding melatonin to replant soil can promote plant growth by improving the rhizosphere soil environment and nitrogen metabolism. In replant soil, chlorophyll synthesis was blocked, reactive oxygen species (ROS) accumulated in large quantities, and membrane lipid peroxidation was aggravated; this eventually resulted in slow plant growth. However, the application of 200 µM exogenous melatonin enhanced the tolerance of plants to ARD by up-regulating the expression of antioxidant enzyme-related genes and increasing ROS scavenging enzyme activity. Exogenous melatonin also increased the absorption and utilization of 15N by increasing the expression of nitrogen absorption genes and the activity of nitrogen metabolism enzymes. Exogenous melatonin enhanced the soil microbial environment by promoting soil enzyme activity and bacterial richness and decreasing the abundance of several harmful fungi in rhizosphere soil. Mantel test results showed that soil properties (except for AP) and growth indexes were positively correlated with the rate of 15N absorption and utilization. Spearman correlation analysis showed that the above factors were closely related to the richness and diversity of bacteria and fungi, indicating that the composition of microbial communities might play a key role in mediating change in the soil environment and thus affect nutrient absorption and growth. These findings provide new insights into how melatonin enhances ARD tolerance.

Improved tolerance of apple plants to drought stress and nitrogen utilization by modulating the rhizosphere microbiome via melatonin and dopamine.

This study explored the contributions of melatonin and dopamine to the uptake and utilization of nitrogen and the formation of rhizosphere microbial communities in 'Tianhong 2'/M. hupehensis, with the goal improving plant resistance to drought stress. Drought stress was formed by artificially controlling soil moisture content. And melatonin or dopamine solutions were applied to the soil at regular intervals for experimental treatment. After 60 days of treatment, plant indices were determined and the structure of the rhizosphere microbial community was evaluated using high-throughput sequencing technology. The findings revealed two ways through which melatonin and dopamine alleviate the inhibition of growth and development caused by drought stress by promoting nitrogen uptake and utilization in plants. First, melatonin and dopamine promote the absorption and utilization of nitrogen under drought stress by directly activating nitrogen transporters and nitrogen metabolism-related enzymes in the plant. Second, they promote the absorption of nitrogen by regulating the abundances of specific microbial populations, thereby accelerating the transformation of the soil nitrogen pool to available nitrogen that can be absorbed directly by plant roots and utilized by plants. These findings provide a new framework for understanding how melatonin and dopamine regulate the uptake and utilization of nitrogen in plants and improve their ability to cope with environmental disturbances.

Melatonin and dopamine mediate the regulation of nitrogen uptake and metabolism at low ammonium levels in Malus hupehensis.

In plants, ammonium (NH4+) is the main nitrogen source and acts as a physiological and morphological response signaling molecule. Melatonin and dopamine are associated with plant responses to abiotic stress. However, previous studies have rarely focused on nutrient stress, and the roles of melatonin and dopamine in the uptake and metabolism of nitrogen in plants remain unclear. In this study, we investigated the regulatory effects of melatonin and dopamine on nitrogen utilization efficiency in apple seedlings under two NH4+ concentrations (2 and 0.1 mM) by measuring plant growth, root system architecture, 15NH4+ content, and related enzyme activity and gene expression. Under low nitrogen supply, apple seedling growth slowed and showed marked reductions in biomass accumulation, chlorophyll content, and nutrient uptake. However, both melatonin and dopamine significantly improved plant growth, chlorophyll content, and root development and enhanced antioxidant enzyme activity. Exogenous application of melatonin or dopamine also promoted the absorption and accumulation of 15NH4+ and enhanced nitrogen metabolism-related enzyme activity. At the molecular level, melatonin and dopamine significantly increased the expression levels of nitrogen metabolism genes and transporter genes. Overall, these results suggest that melatonin and dopamine can relieve nutrient stress caused by low concentrations of NH4+ through regulating the absorption and metabolism of nitrogen.

Preparation of 125I brachytherapy seeds by iodinating carbon bars with a silver coating.

A method for preparing 125I brachytherapy seeds is presented. Carbon bars were used as the substrates, on which a silver coating was deposited by electroless plating, and then, 125I was adsorbed by iodinating the silver coating so as to prepare source cores. The radioactive cores were sealed individually in the titanium capsule to fabricate the 125I brachytherapy seeds. Quality control checking of the encapsulated 125I seeds for dimension, leakage and surface contamination were performed. And the 125I seeds using carbon bar as the core substrate underwent computed tomography (CT) scan and X-ray imaging to determine the visualization. This paper provides valuable experiences and data for the preparation of 125I brachytherapy seeds.

Crop Load Influences Growth and Hormone Changes in the Roots of "Red Fuji" Apple.

Crop load has a substantial impact on growth of the aerial and belowground parts of apple trees. Here, we examined the effects of different crop loads on growth and hormone levels in apple roots. A crop load of 1.5 (T1.5) fruits per cm2 trunk cross-sectional area (TCSA) treatment resulted in lower root growth vigor, while non-fruiting (T0) and T0.4 conditions showed higher root growth vigor. In all treatments, dead roots increased in length 90 days after full bloom (DAFB), whereas live roots were more abundant at about 50 and 170 DAFB, showing a bimodal curve. During each root growth peak, levels of cytokinins (CTKs), indole acetic acid (IAA), and gibberellic acid (GA3) were higher. Moreover, hormone levels gradually decreased with increasing crop load within each peak. Root turnover tended to decrease with decreasing crop load. These findings indicate that root growth and hormone contents were positively correlated during the fruit growth phase, and that the negative impact of crop load on root growth may have been caused by hormone level decreases.

Preparation of (103)Pd brachytherapy seeds by electroless plating of (103)Pd onto carbon bars.

A method for preparing (103)Pd brachytherapy seeds is reported. The key of the method was to deposit (103)Pd onto carbon bars by electroless plating so as to prepare source cores. After each carbon bar with (103)Pd was sealed in a titanium capsule, the (103)Pd seeds were fabricated. This paper provides valuable experiences and data for the preparation of (103)Pd brachytherapy seeds.

BRMS1 Sensitizes Breast Cancer Cells to ATP-Induced Growth Suppression.

Purinergic signaling may represent an effective target in cancer therapy because the expression of purinergic receptors is altered in many forms of cancer and extracellular nucleotides modulate cancer cell growth. We examined the effect of extracellular ATP on the growth of the metastatic breast carcinoma cell line MDA-MB-435 relative to an immortalized breast epithelial cell line, hTERT-HME1. We also investigated whether the metastasis suppressor gene BRMS1 alters the sensitivity of breast cancer cells to ATP. Exposure to ATP for 24 h decreased proliferation and induced apoptosis in hTERT-HME1. However, exposure to ATP did not decrease proliferation or induce apoptosis in MDA-MD-435 cells until 48 h of exposure and only at higher doses than were effective with hTERT-HME1, suggesting MDA-MB-435 cells were resistant to the antiproliferative and apoptosis-inducing effects of ATP. Exposure to ATP for 24 h induced a decrease in proliferation of MDA-MB-435 cells expressing BRMS1, similar to hTERT-HME1, but did not induce an increase in apoptosis. MDA-MB-435 cells expressed low levels of the purinergic receptor P2Y2, as well as decreased ATP-induced cytosolic calcium mobilization, relative to hTERT-HME1. However, expressing BRMS1 in MDA-MB-435 cells restored P2Y2 levels and ATP-induced cytosolic calcium mobilization such that they were similar to hTERT-HME1. These data suggest that BRMS1 increases the sensitivity of breast cancer cells to the antiproliferative, but not apoptosis-inducing effects of ATP and that this is at least partly mediated by increased expression of the P2Y2 receptor.

Age-related changes in gap junctional intercellular communication in osteoblastic cells.

Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions--membrane-spanning channels that allow direct cell-to-cell conductance of small signaling molecules--are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated GJIC. PTH demonstrated age-dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC.

Expressing connexin 43 in breast cancer cells reduces their metastasis to lungs.

Recently the concept that gap junctions play a role in cancer cell metastasis has emerged. However, the mechanism by which this might occur is unknown. To examine this issue a metastatic breast cancer cell line, MDA-MB-435, was stably transfected with human Cx43 cDNA. Four clones of 435 transfectants (435/Cx43(+) c1, c6, c8, c14) and two clones of plasmid control (435/hy) were isolated and examined in this study. We found that expressing Cx43 in MDA-MB-435 cells decreased their expression of Cx32 but did not affect gap junctional intercellular communication, migration or invasion through Matrigel((R)). However, forced expression of Cx43 decreased the growth of MDA-MB-435 cells, decreased expression of N-cadherin, which is frequently associated with an aggressive phenotype, and increased MDA-MB-435 sensitivity to apoptosis. More importantly, there were fewer lung metastases in mice injected with 435/Cx43(+) cells relative to mice injected with 435/hy. These results suggest that expressing Cx43 in breast cancer cells decreases their metastatic potential through a mechanism independent of gap junctional communication but, rather, related to N-cadherin expression and apoptosis.

Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential.

Emerging evidence suggests that gap junctional intercellular communication (GJIC) and expression of connexins (Cx) contribute to the metastatic potential of breast cancer cells. To more directly address this, an aggressive bone metastasis breast cancer cell line, MDA-MET (MET), was stably transfected with human Cx43 cDNA (MET/Cx43(+)). Focusing on clone 28 of MET/Cx43(+), we demonstrated that GJIC, Cx43 protein and Cx43 mRNA were significantly increased in MET/Cx43(+) cells relative to MET, the plasmid control for the Cx43 transfectants (MET/HY) and a metastatic breast cancer cell that is less metastatic to bone than MET, MDA-MB-231. Cx26 mRNA was also increased in MET/Cx43(+ )clone 28 cells while mRNA for Cx32, Cx37, Cx40 and Cx45 were not detected in any of the breast cancer cell lines examined. MET/Cx43(+ )clone 28 invasiveness was decreased by 33% relative to MET/HY, while their ability to migrate was unchanged. The ability of MET/Cx43(+ )clone 28 cells to adhere to hFOB and HUV-EC-C cells was decreased approximately 30% and 70%, respectively, relative to MET and MET/HY. E-cadherin and N-cadherin proteins were not detected in MET, MDA-MB-231, MET/Cx43(+ )clone 28 and MET/HY cells. However, OB-cadherin protein levels were decreased approximately 43% in MET/Cx43(+ )clone 28 relative to MET/HY cells. These findings suggest that GJIC and Cx43 expression contribute to breast cancer cell adhesion and migration, possibly through a mechanism involving OB-cadherin, and these changes in turn regulate the metastatic potential of breast cancer cells, especially to bone.

Connexin 43 gene expression in mice with cardiopulmonary developmental defects.

Gap junctions are vital for cellular integrity, including homeostasis, morphogenesis, differentiation and growth in normal development of organs such as heart. Connexin 43 (Cx43) is a major gap junction protein. Our cDNA microarray analysis of normal and nitrofen-exposed neonatal mice with hypoplastic lungs, associated congenital diaphragmatic hernia (CDH) and heart developmental defects showed up-regulation of Cx43. Our objective was to establish if cardiopulmonary defects in nitrofen-exposed mice may be linked to altered expression of the Cx43 gene. We addressed our objective by performing northern blot analysis, real-time RT-PCR, immunoblotting and immunohistochemistry by localizing Cx43 in hearts and lungs of normal and nitrofen-exposed mice at different gestational stages. The data confirmed up-regulation of Cx43 expression in both hearts and lungs of CDH neonate mice and in lungs at other developmental stages except the pseudoglandular stage. However, Cx43 protein levels were either the same or less in hearts and lungs of nitrofen-exposed mice than in normal tissues except in pseudoglandular lungs. Different expressions of mRNA and protein suggest possible post-transcriptional or translational defects in Cx43. We observed dysmorphic hearts with exaggerated interventricular grooves and deep notches at the apex of the hearts in nitrofen-exposed fetal/neonatal mice; narrowed pulmonary out-flow and various degrees of craniofacial defects in 15-20% of the affected mice. Our data suggest a possible involvement of Cx43 in craniofacial, heart and lung defects in nitrofen-exposed mice. Such cardiopulmonary defects are also observed in human newborns with CDH. Thus, the murine data may help elucidate the pathways of cardiopulmonary defects in the human newborn condition.

Modulation of connexin43 alters expression of osteoblastic differentiation markers.

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43(-)). hFOB/Cx43(-) cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43(-) cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43(-) cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor alpha1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43(-). Osteopontin mRNA levels were increased in hFOB/Cx43(-) relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43(-) and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.

Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics.

Integrin-mediated adhesion of anchorage-dependent cells to scaffolds is a critical component of tissue engineering. We investigated integrin expression by the human fetal osteoblastic cell line, hFOB 1.19 (hFOB), as a function of substratum surface wettability. The influence of surface wettability on bone cell phenotype was also examined. Plasma-treated quartz (PTQ) and glass (PTG) (hydrophilic, contact angles of 0 degrees), octadecyltrichlorosilane-treated quartz (STQ) and glass (STG) (hydrophobic, contact angles above about 100 degrees), and tissue culture polystyrene were used for cell culture. hFOB cells cultured on hydrophilic substrata displayed well-developed actin stress fibers relative to cells on hydrophobic substrata. Western blot analysis revealed that hFOB cells cultured on hydrophobic substrata (STQ or STG) express lower levels of alphav and beta3 integrin subunits than do cells on hydrophilic substrata (PTQ or PTG). This effect was more pronounced in cells on STQ than on STG. These variations in integrin expression were lessened by extended culture time. Double- labeled integrin/actin immunofluorescence confirmed Western blot results, that is, cells cultured on PTQ displayed distinct, large plaques of alphav and beta3 subunits and integrin alphavbeta3, as well as their colocalization with actin stress fiber ends, whereas cells on STQ did not display integrin plaques after 24 h and displayed only minimal plaque formation after 3 days. Vinculin, a focal adhesion protein that mediates binding between the integrin and actin cytoskeleton, appeared in Western blots to mimic the variations of alphav and beta3 expression with respect to surface wettability. Interestingly, real-time RT-PCR analysis showed that hFOB cultured on hydrophobic substrata, which have downregulated alphav and beta3 integrin subunits, displayed greater steady state mRNA levels of osteopontin, an extracellular matrix (ECM) protein containing the Arg-Gly-Asp (RGD) integrin recognition sequence, than did cells cultured on hydrophilic substrata. Our results imply that substratum surface wettability regulates integrin-mediated bone cell adhesion and further influences the expression of bone cell-ECM complexes.

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells.

The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.