El secretoma de los osteocitos estimulados mecánicamente modula la función de las células mesenquimales / The secretome of mechanically stimulated osteocytes modulates the function of mesenchymal cells

El esqueleto es un órgano metabólicamente activo que se remodela continuamente a la largo de nuestra vida. Estaremodelación implica un equilibrio entre la formación de hueso llevada a cabo por los osteoblastos y la resorción porlos osteoclastos. Los osteocitos son los encargados de regular estos dos procesos y su estimulación mecánica, es esen-cial para mantener el buen funcionamiento óseo y prevenir enfermedades como la osteoporosis. La estimulación de lososteocitos provoca una alteración en la producción y secreción de moléculas de señalización que regulan la actividadde los osteoblastos y los osteoclastos. Las células madre mesenquimales han sido propuestas como posible terapiacelular para la regeneración de distintos tejidos, incluido el tejido óseo. Hipotetizamos en el presente estudio que elsecretoma de células osteocíticas de ratón estimuladas mecánicamente afecta a la capacidad proliferativa, adhesiva ya la expresión génica de células mesenquimales indiferenciadas y células mesenquimales preosteoblásticas. Para ello,se analizaron los procesos biológicos mencionados en líneas continuas celulares preosteoblásticas y células mesenqui-males de ratón en presencia del medio condicionado por células osteocíticas MLO-Y4, después de ser sometidas aestímulo mecánico por flujo de fluido. Se observó que la proliferación aumentó en ambas líneas celulares en presenciadel secretoma de osteocitos estimulados mecánicamente frente al control, mientras que osteocitos no mecanoestimu-lados provocaban su disminución. También se observó un aumento en la capacidad adhesiva de células C3H/10T1/2 trasla estimulación con el secretoma de osteocitos mecanoestimulados. En cuanto a la expresión de genes, solo el factoradipogénico PPARᵞ sufrió alteraciones en células MC3T3-E1 por el secretoma de osteocitos. Estos estudios indican quelos osteocitos pueden modificar el comportamiento biológico de células mesenquimales mediante la secreción de factores solubles.(AU)

MINDIN Exerts Protumorigenic Actions on Primary Prostate Tumors via Downregulation of the Scaffold Protein NHERF-1.

Advanced prostate cancer preferential metastasis to bone is associated with osteomimicry. MINDIN is a secreted matrix protein upregulated in prostate tumors that overexpresses bone-related genes during prostate cancer progression. Na+/H+ exchanger regulatory factor (NHERF-1) is a scaffold protein that has been involved both in tumor regulation and osteogenesis. We hypothesize that NHERF-1 modulation is a mechanism used by MINDIN to promote prostate cancer progression. We analyzed the expression of NHERF-1 and MINDIN in human prostate samples and in a premetastatic prostate cancer mouse model, based on the implantation of prostate adenocarcinoma TRAMP-C1 (transgenic adenocarcinoma of the mouse prostate) cells in immunocompetent C57BL/6 mice. The relationship between NHERF-1 and MINDIN and their effects on cell proliferation, migration, survival and osteomimicry were evaluated. Upregulation of MINDIN and downregulation of NHERF-1 expression were observed both in human prostate cancer samples and in the TRAMP-C1 model. MINDIN silencing restored NHERF-1 expression to control levels in the mouse model. Stimulation with MINDIN reduced NHERF-1 expression and triggered its mobilization from the plasma membrane to the cytoplasm in TRAMP-C1 cells. MINDIN-dependent downregulation of NHERF-1 promoted tumor cell migration and proliferation without affecting osteomimicry and adhesion. We propose that MINDIN downregulates NHERF-1 expression leading to promotion of processes involved in prostate cancer progression.

MINDIN secretion by prostate tumors induces premetastatic changes in bone via ß-catenin.

Bone metastases are common in advanced prostate cancer patients, but mechanisms by which specific pro-metastatic skeletal niches are formed before tumor cell homing are unclear. We aimed to analyze the effects of proteins secreted by primary prostate tumors on the bone microenvironment before the settlement and propagation of metastases. Here, using an in vivo pre-metastatic prostate cancer model based on the implantation of prostate adenocarcinoma TRAMP-C1 cells in immunocompetent C57BL/6 mice, we identify MINDIN as a prostate tumor secreted protein that induces bone microstructural and bone remodeling gene expression changes before tumor cell homing. Associated with these changes, increased tumor cell adhesion to the endosteum ex vivo and to osteoblasts in vitro was observed. Furthermore, MINDIN promoted osteoblast proliferation and mineralization and monocyte expression of osteoclast markers. ß-catenin signaling pathway revealed to mediate MINDIN actions on osteoblast gene expression but failed to affect MINDIN-induced adhesion to prostate tumor cells or monocyte differentiation to osteoclasts. Our study evidences that MINDIN secretion by primary prostate tumors creates a favorable bone environment for tumor cell homing before metastatic spread.

Role of Calcium Signaling in Prostate Cancer Progression: Effects on Cancer Hallmarks and Bone Metastatic Mechanisms.

Advanced prostate cancers that progress to tumor metastases are often considered incurable or difficult to treat. The etiology of prostate cancers is multi-factorial. Among other factors, de-regulation of calcium signals in prostate tumor cells mediates several pathological dysfunctions associated with tumor progression. Calcium plays a relevant role on tumor cell death, proliferation, motility-invasion and tumor metastasis. Calcium controls molecular factors and signaling pathways involved in the development of prostate cancer and its progression. Such factors and pathways include calcium channels and calcium-binding proteins. Nevertheless, the involvement of calcium signaling on prostate cancer predisposition for bone tropism has been relatively unexplored. In this regard, a diversity of mechanisms triggers transient accumulation of intracellular calcium in prostate cancer cells, potentially favoring bone metastases development. New therapies for the treatment of prostate cancer include compounds characterized by potent and specific actions that target calcium channels/transporters or pumps. These novel drugs for prostate cancer treatment encompass calcium-ATPase inhibitors, voltage-gated calcium channel inhibitors, transient receptor potential (TRP) channel regulators or Orai inhibitors. This review details the latest results that have evaluated the relationship between calcium signaling and progression of prostate cancer, as well as potential therapies aiming to modulate calcium signaling in prostate tumor progression.

The secreted matrix protein mindin increases prostate tumor progression and tumor-bone crosstalk via ERK 1/2 regulation.

Advanced prostate cancer cells preferentially metastasize to bone by acquiring a bone phenotype that allows metastatic cells to thrive in the skeletal environment. Identification of factors that promote the expression of ectopic bone genes-process known as osteomimicry-leading to tumor progression is crucial to prevent and treat metastatic prostate cancer and prolong life expectancy for patients. Here, we identify the extracelular matrix protein mindin in the secretome of prostate adenocarcinoma cells and show that mindin overexpression in human and mouse TRAMP-C1-induced prostate tumors correlates with upregulated levels of bone-related genes in the tumorigenic prostate tissues. Moreover, mindin silencing decreased osteomimicry in adenocarcinoma cells and in the prostate tumor mice model, as well as reduced tumor cell proliferation, migration and adhesion to bone cells. Inhibition of the extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation decreased the proliferative, migratory and pro-adhesion actions of mindin on prostate tumor cells. In addition, conditioned media obtained by crosstalk stimulation of either osteocytes or osteoblasts with the secretome of TRAMP-C1 cells promoted osteomimicry in prostate tumor cells; an effect inhibited by mindin silencing of TRAMP-C1 cells. In vivo, tibiae of primary tumor-bearing mice overexpressed the pro-angiogenic and pro-metastattic factor vascular endothelial growth factor receptor 2 (VEGFR2) in a mindin-dependent manner. Our findings indicate that mindin is a novel regulator of osteomimicry in prostate tumors and potentially mediates tumor-bone cell crosstalk, suggesting its promising role as a target to inhibit bone metastases.

Local delivery of parathyroid hormone-related protein-derived peptides coated onto a hydroxyapatite-based implant enhances bone regeneration in old and diabetic rats.

Diabetes mellitus (DM) and aging are associated with bone fragility and increased fracture risk. Both (1-37) N- and (107-111) C-terminal parathyroid hormone-related protein (PTHrP) exhibit osteogenic properties. We here aimed to evaluate and compare the efficacy of either PTHrP (1-37) or PTHrP (107-111) loaded into gelatin-glutaraldehyde-coated hydroxyapatite (HA-Gel) foams to improve bone repair of a transcortical tibial defect in aging rats with or without DM, induced by streptozotocin injection at birth. Diabetic old rats showed bone structural deterioration compared to their age-matched controls. Histological and µ-computerized tomography studies showed incomplete bone repair at 4 weeks after implantation of unloaded Ha-Gel foams in the transcortical tibial defects, mainly in old rats with DM. However, enhanced defect healing, as shown by an increase of bone volume/tissue volume and trabecular and cortical thickness and decreased trabecular separation, occurred in the presence of either PTHrP peptide in the implants in old rats with or without DM. This was accompanied by newly formed bone tissue around the osteointegrated HA-Gel implant and increased gene expression of osteocalcin and vascular endothelial growth factor (bone formation and angiogenic markers, respectively), and decreased expression of Sost gene, a negative regulator of bone formation, in the healing bone area. Our findings suggest that local delivery of PTHrP (1-37) or PTHrP (107-111) from a degradable implant is an attractive strategy to improve bone regeneration in aged and diabetic subjects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2060-2070, 2016.

Adverse Effects of Diabetes Mellitus on the Skeleton of Aging Mice.

In the present study, the possibility that a diabetic (DM) status might worsen age-related bone deterioration was explored in mice. Male CD-1 mice aged 2 (young control group) or 16 months, nondiabetic or made diabetic by streptozotocin injections, were used. DM induced a decrease in bone volume, trabecular number, and eroded surface, and in mineral apposition and bone formation rates, but an increased trabecular separation, in L1-L3 vertebrae of aged mice. Three-point bending and reference point indentation tests showed slight changes pointing to increased frailty and brittleness in the mouse tibia of diabetic old mice. DM was related to a decreased expression of both vascular endothelial growth factor and its receptor 2, which paralleled that of femoral vasculature, and increased expression of the pro-adipogenic gene peroxisome proliferator-activated receptor γ and adipocyte number, without affecting ß-catenin pathway in old mouse bone. Concomitant DM in old mice failed to affect total glutathione levels or activity of main anti-oxidative stress enzymes, although xanthine oxidase was slightly increased, in the bone marrow, but increased the senescence marker caveolin-1 gene. In conclusion, DM worsens bone alterations of aged mice, related to decreased bone turnover and bone vasculature and increased senescence, independently of the anti-oxidative stress machinery.

The vertebrae of prematurely aging mice as a skeletal model of involutional osteoporosis.

Oxidative stress in bone increases with age, which leads to bone frailty and a high fracture risk. Animal models show that early changes in trabecular structure occur in age-related osteopenia. These models might be valuable to assess the contribution of oxidative stress in age-related bone loss. Premature aging mice (PAM) have previously been characterized as a model of premature immunological and neurological senescence. PAM long bones (mainly consisting of cortical bone) display features of aging bone. Thus, we aimed to evaluate the vertebrae, representing a unique poorly loaded type of trabecular bone in mice, in PAM and no PAM (NPAM) controls. PAM showed an anxious behaviour, based on physical activity evaluation. These mice had decreased bone mineral density (0.078 mg/cm² in NPAM vs 0.070 g/cm² in PAM; p⟨0.05); a decreased number of osteocytes per bone field (404±36 in NPAM vs 320±27 in PAM; p⟨0.01); and downregulation of various osteoblastic genes and low eroded surface/bone surface, 4.2±0.5 in NPAM vs 1.9±0.2 in PAM; p⟨0.01). This was associated with increased expression of oxidative stress markers, Foxo1 and GADD45, in PAM vertebrae. Mesenchymal progenitors in the bone marrow of PAM have a poor mineralization capacity (assessed by the number of mineralized nodules and suface), and showed a lower response to an osteogenic input -represented by parathormone-related protein-, compared to NPAM. Collectively, these results indicate that PAM vertebrae show osteopenia related to diminished bone formation and remodeling. Our findings further support the validity of PAM as a suitable model for involutional osteoporosis and its treatment.

Normalizing action of exendin-4 and GLP-1 in the glucose metabolism of extrapancreatic tissues in insulin-resistant and type 2 diabetic states.

Exendin-4 (Ex-4) mimics glucagon-like peptide-1 (GLP-1 or GCG as listed in the HUGO database), being anti-diabetic and anorectic, in stimulating glucose and lipid metabolism in extrapancreatic tissues. We studied the characteristics of Ex-4 and GLP-1 action, during prolonged treatment, on GLUTs expression (mRNA and protein), glycogen content (GC), glucose transport (GT), glycogen synthase a (GSa), and kinase (PI3K and MAPKs) activity, in liver, muscle, and fat of insulin-resistant (IR, by fructose) and type 2 diabetic (T2D, streptozotocin at birth) rats compared with normal rats. In both IR and T2D, the three tissues studied presented alterations in all measured parameters. In liver, GLP-1 and also Ex-4 normalized the lower than normal Glut2 (Slc2a2) expression and showed a trend to normalize the reduced GC in IR, and GLP-1, like Ex-4, also in T2D, effects mediated by PI3K and MAPKs. In skeletal muscle, neither GLP-1 nor Ex-4 modified Glut4 (Slc2a4) expression in either experimental model but showed normalization of reduced GT and GSa, in parallel with the normalization of reduced PI3K activity in T2D and MAPKs in both models. In adipose tissue, the altered GLUT4 expression in IR and T2D, along with reduced GT in IR and increased GT in T2D, and with hyperactivated PI3K in both, became normal after GLP-1 and Ex-4 treatment; yet, MAPKs, that were also higher, became normal only after Ex-4 treatment. The data shows that Ex-4, as well as GLP-1, exerts a normalizing effect on IR and T2D states through a distinct post-receptor mechanism, the liver being the main target for Ex-4 and GLP-1 to control glucose homeostasis.

GLP-1 and exendin-4 can reverse hyperlipidic-related osteopenia.

Increased fat mass contributes to bone deterioration. Glucagon-like peptide 1 (GLP-1) and its related peptide exendin 1-39 amide (Ex-4), two lipid-lowering peptides, exert osteogenic effects in diabetic states. We examined the actions of 3-day administration of GLP-1 or Ex-4 on bone remodeling markers and on bone mass and structure in hyperlipidic (HL) and hypercaloric rats. Wistar rats on a hyperlipidemic diet for 35 days were subcutaneously administered GLP-1 (0.86  nmol/kg per h), Ex-4 (0.1  nmol/kg per h), or saline (control) by continuous infusion for 3 days. After killing, tibiae were removed for total RNA and protein isolation, as well as femurs and L1-L4 vertebrae for bone mass and quality assessment. Body weight and plasma insulin were unaltered in HL rats, which showed osteopenia (by dual-energy X-ray absorptiometry), associated with hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. GLP-1 or Ex-4 administration decreased the levels of glucose, triglycerides, and total cholesterol in plasma but increased osteocalcin (OC) gene expression and the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) ratio - at the expense of an augmented OPG - above corresponding control values in the tibia. Each tested peptide similarly reversed the decreased femoral and vertebral bone mass in these rats, whereas the deteriorated trabecular structure in the vertebrae improved associated with normalization of bone remodeling. These findings demonstrate that GLP-1 and Ex-4 are similarly efficient in reversing the bone alterations in this HL rat model, which has proven to be useful for studying the fat-bone relationships.

Characteristic of GLP-1 effects on glucose metabolism in human skeletal muscle from obese patients.

Direct effects of GLP-1, kinase-mediated, on glucose and lipid metabolism in rat and human extrapancreatic tissues, are amply documented and also changes in type-2 diabetic (T2D) patients. Here, we explored the characteristics of the GLP-1 action and those of its analogs Ex-4 and Ex-9, on muscle glucose transport (GT) and metabolism in human morbid obesity (OB), as compared with normal and T2D subjects. In primary cultured myocytes from OB, GT and glycogen synthase a (GSa) activity values were lower than normal, and comparable to those reported in T2D patients; GT was increased by either GLP-1 or Ex-9 in a more efficient manner than in normal or T2D, up to normal levels; the Ex-4 increasing effect on GSa activity was two times that in normal cells, while Ex-9 failed to modify the enzyme activity. In OB, the control value of all kinases analyzed - PI3K, PKB, MAPKs, and p70s6K - although lower than that in normal or T2D subjects, the cells maintained their response capability to GLP-1, Ex-4, Ex-9 and insulin, with some exceptions. GLP-1 and exendins showed a direct normalizing action in the altered glucose uptake and metabolism in the muscle of obese subjects, which in the case of GLP-1 could account, at least in part, for the reported restoration of the metabolic conditions of these patients after restrictive surgery.