Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

The effects of acute hyperinsulinemia on bone metabolism.

Insulin signaling in bone-forming osteoblasts stimulates bone formation and promotes the release of osteocalcin (OC) in mice. Only a few studies have assessed the direct effect of insulin on bone metabolism in humans. Here, we studied markers of bone metabolism in response to acute hyperinsulinemia in men and women. Thirty-three subjects from three separate cohorts (n=8, n=12 and n=13) participated in a euglycaemic hyperinsulinemic clamp study. Blood samples were collected before and at the end of infusions to determine the markers of bone formation (PINP, total OC, uncarboxylated form of OC (ucOC)) and resorption (CTX, TRAcP5b). During 4 h insulin infusion (40 mU/m(2) per min, low insulin), CTX level decreased by 11% (P<0.05). High insulin infusion rate (72 mU/m(2) per min) for 4 h resulted in more pronounced decrease (-32%, P<0.01) whereas shorter insulin exposure (40 mU/m(2) per min for 2 h) had no effect (P=0.61). Markers of osteoblast activity remained unchanged during 4 h insulin, but the ratio of uncarboxylated-to-total OC decreased in response to insulin (P<0.05 and P<0.01 for low and high insulin for 4 h respectively). During 2 h low insulin infusion, both total OC and ucOC decreased significantly (P<0.01 for both). In conclusion, insulin decreases bone resorption and circulating levels of total OC and ucOC. Insulin has direct effects on bone metabolism in humans and changes in the circulating levels of bone markers can be seen within a few hours after administration of insulin.

Novel perspectives on the transcytotic route in osteoclasts.

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

Serum osteocalcin is not associated with glucose but is inversely associated with leptin across generations of nondiabetic women.

CONTEXT: The skeleton is recognized as an important player in energy metabolism through its interactions with other tissues. Whether the association of osteocalcin with glucose metabolism is age dependent has not been fully addressed. OBJECTIVE: The objective of the study was to examine the age-specific association between different forms of osteocalcin and glucose and adipokines. DESIGN: This was a family-based study across three generations. SETTING: The study was conducted at a university laboratory. PARTICIPANTS: Sixty-four daughter-premenopausal mother-maternal grandmother trios participated in the study. METHODS: Fasting plasma glucose and insulin concentrations, serum total (tOC), carboxylated (cOC), and uncarboxylated (ucOC = tOC - cOC) osteocalcin, leptin, and adiponectin levels, and fat masses were assessed. Generalized estimating equations (GEE) model was used to assess the associations of bone biomarkers with glucose metabolism variables and adipokines. RESULTS: No significant difference in insulin was found between generations, whereas glucose and leptin increased with age. Levels of tOC, cOC, and ucOC were highest in girls and lowest in mothers (P < 0.01). Grandmothers had higher leptin and adiponectin than mothers and girls. Despite the differences in insulin and glucose between the low and high homeostasis model assessment insulin resistance index (HOMA-IR) groups within generations, no significant differences in tOC, cOC, and ucOC were found. Compared with their low HOMA-IR counterparts, the high HOMA-IR group had significantly higher leptin and lower adiponectin in mothers and grandmothers. The tOC, cOC, and ucOC levels did not correlate with HOMA-IR, leptin, or adiponectin when the three generations were evaluated together, but when separated by generation, leptin was inversely correlated with tOC (P = 0.003) and cOC (P = 0.047) in mothers and with ucOC in grandmothers (P = 0.042). CONCLUSIONS: Osteocalcin, glucose, and adipokines change with age but in a noncommensurate manner. We infer that the association between osteocalcin and glucose metabolism is minor and age specific in nondiabetic women. Leptin, however, strongly correlated with insulin resistance independently of fat masses, suggesting that obesity, as a metabolic disorder risk factor, affects glucose metabolism, partly through the role of leptin.

Rab13 is upregulated during osteoclast differentiation and associates with small vesicles revealing polarized distribution in resorbing cells.

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.

Urinary osteocalcin and serum pro-C-type natriuretic peptide predict linear catch-up growth in infants.

Preterm (PT) infants are at risk of growth failure despite advanced early care and nutrition. In addition to poor weight gain, slow postnatal linear growth also is associated with adverse neurological outcome. Markers distinguishing infants at risk for impaired catch-up growth are needed. The aim of this longitudinal study was to determine the extent to which postnatal levels of circulating cartilage (serum pro-C-type natriuretic peptide [S-proCNP]) and urinary bone metabolic markers (urinary osteocalcin [MidOC] and two forms of C-terminal cross-linked telopeptide of type I collagen [U-α-CTX-I and U-ß-CTX-I]) predict catch-up growth in infancy in 67 PT and 58 full-term (FT) infants. PT infants were significantly shorter than FT infants during the first 6 months of life, but no statistically significant difference was found at the corrected age of 14 months (M14). At the age of 3 months (M3), S-ProCNP and U-MidOC levels, but not U-α-CTX-I and U-ß-CTX-I levels, correlated positively with prospective growth velocity from M3 to M14 (ρ = 0.460, p < 0.001 and ρ = 0.710, p < 0.001, respectively). In predicting slow linear growth (growth velocity at the lowest quartile), the area under the S-ProCNP ROC curve was 0.662 and that of U-MidOC 0.891. Thus, U-MidOC, and to lesser extent S-ProCNP at M3 are predictors of catch-up growth in infancy.

Transient 100 nM dexamethasone treatment reduces inter- and intraindividual variations in osteoblastic differentiation of bone marrow-derived human mesenchymal stem cells.

The development of in vitro culturing techniques for osteoblastic differentiation of human mesenchymal stem cells (hMSC) is important for cell biology research and the development of tissue-engineering applications. Dexamethasone (Dex) is a commonly used supplement, but the optimal use of Dex treatment is still unclear. By adjusting the timing of Dex supplementation, the negative effects of long-term Dex treatment could be overcome. Transient Dex treatment could contribute toward minimizing broad donor variation, which is a major challenge. We compared the two most widely used Dex concentrations of 10 and 100 nM as transient or continuous treatment and studied inter- and intraindividual variations in osteoblastic differentiation of hMSC. Characterized bone marrow-derived hMSC from 17 female donors of different age groups were used. During osteoblastic induction, the cells were treated with 10 or 100 nM Dex either transiently for different time periods or continuously. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Cell proliferation, cell viability, and apoptosis were also monitored. The strongest osteoblastic differentiation was observed when 100 nM Dex was present for the first week. In terms of inter- and intraindividual coefficients of variations, transient treatment with 100 nM Dex was superior to the other culture conditions and showed the lowest variations in all age groups. This study demonstrates that the temporary presence of 100 nM Dex during the first week of induction culture promotes hMSC osteoblastic differentiation and reduces inter- and intraindividual variations. With this protocol, we can reproducibly produce functional osteoblasts in vitro from the hMSC of different donor populations.

The effect of oral glucose tolerance test on serum osteocalcin and bone turnover markers in young adults.

Osteocalcin (OC) is an osteoblast-derived protein implicated in the regulation of glucose tolerance and energy metabolism. This endocrine function has been suggested to be exerted via its undercarboxylated form, which has been shown to induce expression of adiponectin, insulin, and islet cell proliferation in mice. Furthermore, insulin has recently been shown to regulate the biological activity of OC in bone. Our aim was to explore the association between glucose and bone metabolism by evaluating the effect of a standard 75 g oral glucose tolerance test (OGTT) on serum OC, carboxylated OC (cOC) and bone-turnover markers (BTMs) C terminal telopeptide (ßCTX-I) and N terminal propeptide (PINP) of type I collagen and tartrate-resistant acid phosphatase 5b (TRACP5b). Serum samples collected at 0 and at 120 min were analyzed in a cohort of normoglycemic young adults (n = 23, mean age 23.6 years). During OGTT a significant decrease was observed in all BTMs (P < 0.001 for all variables). The median decreases from 0 to 120 min for OC, cOC, ßCTX-I, PINP, and TRACP5b were -32.1% (-37.9 to -19.6), -34.4% (-39.8 to -22.2), -61.4% (-68.5 to -53.0), -26.8% (-33.2 to -19.2), and -44.5% (-48.3 to -40.2), respectively. A strong association between the changes in OC and cOC was observed (r = 0.83, P < 0.001). The decrease in PINP was associated with changes in OC, whereas the changes in ßCTX-I and TRACP5b were not associated with decreases in OC or cOC. The observed OGTT-induced changes in bone-derived proteins were partially independent of each other and potentially mediated by different mechanisms.

Receptor tyrosine kinase inhibition causes simultaneous bone loss and excess bone formation within growing bone in rats.

During postnatal skeletal growth, adaptation to mechanical loading leads to cellular activities at the growth plate. It has recently become evident that bone forming and bone resorbing cells are affected by the receptor tyrosine kinase (RTK) inhibitor imatinib mesylate (STI571, Gleevec®). Imatinib targets PDGF, ABL-related gene, c-Abl, c-Kit and c-Fms receptors, many of which have multiple functions in the bone microenvironment. We therefore studied the effects of imatinib in growing bone. Young rats were exposed to imatinib (150mg/kg on postnatal days 5-7, or 100mg/kg on postnatal days 5-13), and the effects of RTK inhibition on bone physiology were studied after 8 and 70days (3-day treatment), or after 14days (9-day treatment). X-ray imaging, computer tomography, histomorphometry, RNA analysis and immunohistochemistry were used to evaluate bone modeling and remodeling in vivo. Imatinib treatment eliminated osteoclasts from the metaphyseal osteochondral junction at 8 and 14days. This led to a resorption arrest at the growth plate, but also increased bone apposition by osteoblasts, thus resulting in local osteopetrosis at the osteochondral junction. The impaired bone remodelation observed on day 8 remained significant until adulthood. Within the same bone, increased osteoclast activity, leading to bone loss, was observed at distal bone trabeculae on days 8 and 14. Peripheral quantitative computer tomography (pQCT) and micro-CT analysis confirmed that, at the osteochondral junction, imatinib shifted the balance from bone resorption towards bone formation, thereby altering bone modeling. At distal trabecular bone, in turn, the balance was turned towards bone resorption, leading to bone loss.

The critical role of IL-34 in osteoclastogenesis.

It has been widely believed that the cytokines required for osteoclast formation are M-CSF (also known as CSF-1) and RANKL. Recently, a novel cytokine, designated IL-34, has been identified as another ligand of CSF1R. This study was to explore the biological function, specifically osteoclastogenesis and bone metabolism, of the new cytokine. We produced recombinant mouse IL-34 and found that together with RANKL it induces the formation of osteoclasts both from splenocytes as well as dose-dependently from bone marrow cells in mouse and these cells also revealed bone resorption activity. It also promotes osteoclast differentiation from human peripheral blood mononucleated cells. Finally, we show that systemic administration of IL-34 to mice increases the proportion of CD11b+ cells and reduces trabecular bone mass. Our data indicate that IL-34 is another important player in osteoclastogenesis and thus may have a role in bone diseases. Strategies of targeting CSF1/CSF1R have been developed and some of them are already in preclinical and clinical studies for treatment of inflammatory diseases. Our results strongly suggest the need to revisit these strategies as they may provide a new potential pharmaceutical target for the regulation of bone metabolism in addition to their role in the treatment of inflammatory diseases.

Imbalance of the autonomic nervous system at night in women with gestational diabetes.

AIMS: Autonomic nervous system dysfunction is observed in Type 2 diabetes. As gestational diabetes is a potent risk factor of later Type 2 diabetes, we set out to determine whether autonomic nervous system imbalance could already be observed in women with this condition. Because activity of the sympathetic nervous system tends to be relatively stable in the nocturnal hours, we performed the study at night. RESEARCH DESIGN AND METHODS: We studied 41 women with gestational diabetes, 22 healthy pregnant controls and 14 non-pregnant controls. We assayed plasma noradrenaline at 24.00, 04.00 and 07.00 h and performed an overnight Holter recording for heart rate variability analysis. In addition, we assayed plasma adrenomedullin, a cardiovascular protective hormone. RESULTS: Compared with non-pregnant controls, plasma noradrenaline levels were increased at 04.00 and 07.00 h in the gestational diabetic (P = 0.003) and pregnant control (P = 0.002) groups, with no difference between them. Heart rate variability, very-low-frequency and low-frequency power were lower in pregnant groups compared to the non-pregnant controls. Heart rate variability remained unchanged between specified sampling times in the gestational diabetic group, in contrast to fluctuation seen in the control groups. CONCLUSIONS: Gestational diabetes, compared with normal pregnancy, seems not to be a state of overall sympathetic nervous system activation. At the heart level, however, an inhibitory effect on autonomic nervous system modulation was seen. Plasma noradrenaline and heart rate variability correlated well, supporting the use of this function in future studies of overall sympathetic activity during pregnancy.

A rationale for osteoclast selectivity of inhibiting the lysosomal V-ATPase a3 isoform.

Osteoclastic bone resorption can be completely abolished by inhibiting the vacuolar H(+)-ATPase (V-ATPase), a proton pump composed of at least 12 different subunits. However, V-ATPases are ubiquitous and it is unclear whether the osteoclast V-ATPase has a unique composition that would allow its selective inhibition. Aiming to answer this question, we compared human osteoclasts and monocytic THP.1 cells with respect to the localization of the a3 isoform of the 116-kDa subunit, which is indispensable for bone resorption, and sensitivity to SB242784, a V-ATPase inhibitor that prevents experimentally induced osteoporosis. By immunofluorescence, a3 was essentially nondetectable in THP.1 cells, while in osteoclasts a3 was highly upregulated and localized to lysosomes in nonresorbing osteoclasts. We isolated the lysosomal compartment from both sources as latex bead-containing phagolysosomes and compared them. Osteoclast phagolysosomes and THP.1 phagolysosomes both contained a3 and a1; however, the a3/a1 ratio was 3.8- to 11.2-fold higher in osteoclast phagolysosomes. Importantly, the V-ATPase-dependent acidification of phagolysosomes from both sources was essentially equally sensitive to SB242784. Thus, we observed no indication of a qualitative uniqueness of the osteoclast V-ATPase; rather, the high a3-level in osteoclasts may represent an upregulation of the common lysosomal V-ATPase. Our results, together with the reported phenotype of a3 deficiency and the reported efficacy of SB242784 in vivo, suggest that V-ATPase structure-independent mechanisms render bone resorption more sensitive than lysosomal function to V-ATPase inhibition. One such mechanism may be compensation of a3 by a1, which may be sufficient for retaining lysosomal function but not bone resorption.

Inhibition of GGTase-I and FTase disrupts cytoskeletal organization of human PC-3 prostate cancer cells.

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.

Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1.

Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.

Bone turnover markers and prediction of fracture: a prospective follow-up study of 1040 elderly women for a mean of 9 years.

Osteoporosis is characterized by compromised bone mass and strength, predisposing to an increased risk of fracture. Increased bone metabolism has been suggested to be a risk factor for fracture. The aim of this study was to evaluate whether baseline bone turnover markers are associated with long-term incidence of fracture in a population-based sample of 1040 women who were 75 years old (Malmö OPRA study). Seven bone markers (S-TRACP5b, S-CTX-I, S-OC[1-49], S-TotalOC, S-cOC, S-boneALP, and urinary osteocalcin) were measured at baseline and 1-year follow-up visit. During the mean follow-up of 9.0 years (range 7.4-10.9), 363 women sustained at least one fracture of any type, including 116 hip fractures and 103 clinical vertebral fractures. High S-TRACP5b and S-CTX-I levels were associated with increased risk of any fracture with hazard ratios [HRs (95% confidence interval)] of 1.16 (1.04-1.29) and 1.13 (1.01-1.27) per SD increase, respectively. They also were associated with increased risk of clinical vertebral fracture with HRs of 1.22 (1.01-1.48) and 1.32 (1.05-1.67), respectively. Markers were not associated with risk for hip fracture. Results were similar when we used resorption markers, including urinary osteocalcin, measured at the 1-year visit or an average of the two measurements. The HRs were highest for any fracture in the beginning of the follow-up period, 2.5 years from baseline. For vertebral fractures, the association was more pronounced and lasted for a longer period of time, at least for 5 years. In conclusion, elevated levels of S-TRACP5b, S-CTX-I, and urinary osteocalcin are associated with increased fracture risk for up to a decade in elderly women.

Overexpression of vascular endothelial growth factor C increases growth and alters the metastatic pattern of orthotopic PC-3 prostate tumors.

BACKGROUND: Prostate cancer metastasizes to regional lymph nodes and distant sites but the roles of lymphatic and hematogenous pathways in metastasis are not fully understood. METHODS: We studied the roles of VEGF-C and VEGFR3 in prostate cancer metastasis by blocking VEGFR3 using intravenous adenovirus-delivered VEGFR3-Ig fusion protein (VEGFR3-Ig) and by ectopic expression of VEGF-C in PC-3 prostate tumors in nude mice. RESULTS: VEGFR3-Ig decreased the density of lymphatic capillaries in orthotopic PC-3 tumors (p < 0.05) and inhibited metastasis to iliac and sacral lymph nodes. In addition, tumor volumes were smaller in the VEGFR3-Ig-treated group compared with the control group (p < 0.05). Transfection of PC-3 cells with the VEGF-C gene led to a high level of 29/31 kD VEGF-C expression in PC-3 cells. The size of orthotopic and subcutaneous PC-3/VEGF-C tumors was significantly greater than that of PC-3/mock tumors (both p < 0.001). Interestingly, while most orthotopic PC-3 and PC-3/mock tumors grown for 4 weeks metastasized to prostate-draining lymph nodes, orthotopic PC-3/VEGF-C tumors primarily metastasized to the lungs. PC-3/VEGF-C tumors showed highly angiogenic morphology with an increased density of blood capillaries compared with PC-3/mock tumors (p < 0.001). CONCLUSION: The data suggest that even though VEGF-C/VEGFR3 pathway is primarily required for lymphangiogenesis and lymphatic metastasis, an increased level of VEGF-C can also stimulate angiogenesis, which is associated with growth of orthotopic prostate tumors and a switch from a primary pattern of lymph node metastasis to an increased proportion of metastases at distant sites.

Osteocyte-derived HB-GAM (pleiotrophin) is associated with bone formation and mechanical loading.

HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and mu-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.

FGF-8b induces growth and rich vascularization in an orthotopic PC-3 model of prostate cancer.

Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis.

Evidence for the role of osteocytes in the initiation of targeted remodeling.

Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. Even though there is increasing evidence that osteocyte density, microcracks and targeted remodeling are related, there still exist several questions. For example, how osteoclasts are targeted to the specific site of microdamage for repair. It has been proposed that apoptotic osteocytes could secrete a specific signal to target osteoclasts. The other question is the nature of this signal. To elucidate the role of microdamage-induced osteocyte cell death in the initiation of targeted remodelling, this paper discusses the potential use of an in vitro model, in which osteocytes can be three-dimensionally cultured and locally damaged. Furthermore, the method enables one to study the osteocyte-derived soluble interactions with bone marrow cells. It was demonstrated that damaged osteocytes locally affect osteoclast precursors by secreting osteoclastogenic factors, and thus can have a role in the initiation of resorption in bone remodelling. This strongly supports the idea that damage to osteocyte cellular network has the potential to stimulate osteoclastic proliferation and therefore the activation of Basic Multicellular Units (BMUs).