Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1-100 ng/ml, 2-5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 g/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1-28 days, or a transient rhFGF-2 treatment for 1-7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8 -28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8 -28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3-4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor ␤2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation. (J Bone Miner Res 1998;13:645-654)

In summary, the improvement in the formation/resorption marker ratio suggests beneficial systemic and local bone effects of infliximab in patients with RA.

The updated recommendations place strong emphasis on the treatment of women with severe fractures, in whom the use of osteoporosis medications is recommended. All the available osteoporosis medications are suitable in patients with severe fractures; zoledronic acid deserves preference as the fist-line drug after a hip fracture. In patients with or without non-severe fractures, the decision to use osteoporosis medications is based on bone mineral density values and in challenging cases, on probabilities supplied by prediction tools such as FRAX. All osteoporosis medications are suitable; raloxifene should be reserved for patients at low risk for peripheral fractures. The fracture risk should be reevaluated every 2 to 3 years to decide on the best follow-up treatment. These updated recommendations discuss the selection of first-line osteoporosis medications and treatment sequences.

For decades, cancer was associated with gap-junction defects. However, more recently it appeared that the gap junction proteins (connexins) could be re-expressed and participate to cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer (PCa) are known to be connexin deficient, it was interesting to verify whether their bone-targeted metastatic behaviour could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates to the differentiation of osteoblastic cells. Thus, we investigated the effect of the increased Cx43 expression, by retroviral infection, on the metastatic behaviour of two well-characterized cell lines (PC-3 and LNCaP) representing different stages of PCa progression. It appeared that Cx43 differently behaved in those cell lines and induced different phenotypes. In LNCaP, Cx43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those Cx43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of Cx43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of Cx43 may govern the ability of PCa cells to metastasize in bones.

No abstract

Fibroblast growth factors (FGFs) are important factors regulating osteogenesis. However, the early mechanisms and signaling pathways involved in FGF actions in osteoblasts are unknown. We investigated the effects of FGF-2 on cell-cell adhesion and cadherin expression and the underlying signaling pathways in immortalized human neonatal calvaria (IHNC) cells. These cells express E- and N-cadherins, as shown by immunocytochemical and Western blot analyses. rhFGF-2 increased cell-cell adhesion at 24-72 h, as measured in a cell aggregation assay, and this effect was blocked by specific neutralizing anti-N-cadherin, but not anti-E-cadherin antibodies. Accordingly, ELISA and Western blot analyses showed that rhFGF-2 (10-100 ng/ml) dose dependently increased N-cadherin but not E-cadherin protein levels. RT-PCR analysis showed that rhFGF-2 transiently increased N-cadherin mRNA levels in IHNC cells. The RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole prevented the rhFGF-2-induced up-regulation of N-cadherin mRNA, suggesting that transcription is necessary for this effect. Analysis of signaling molecules showed evidence that PLCgamma-PKC, Src, Erk 1/2 and p38 MAPK pathways are activated by rhFGF-2 in IHNC cells. The selective PKC inhibitors calphostin C, Ro-31-8220, Gö6976 and Gö6983 abrogated the stimulatory effect of rhFGF-2 on N-cadherin mRNA levels. The src-family tyrosine kinase inhibitor PP1 also blocked rhFGF-2-promoted N-cadherin expression. In contrast, the p38 MAP kinase inhibitor SB 203580 or the MEK inhibitor PD98059 had no effect on rhFGF-2-induced N-cadherin mRNA levels. Our data indicate that FGF-2 increases N-cadherin expression and function in human calvaria osteoblasts via activation of PKC and src-kinase pathways. This study identifies N-cadherin as a previously unrecognized target gene for FGF-2 signaling pathway that regulates cell-cell adhesion in human osteoblasts.

No abstract

Hypophosphatasia (HPP) is a rare inherited skeletal dysplasia due to loss of function mutations in the ALPL gene. The disease is subject to an extremely high clinical heterogeneity ranging from a perinatal lethal form to odontohypophosphatasia affecting only teeth. Up to now genetic diagnosis of HPP is performed by sequencing the ALPL gene by Sanger methodology. Osteogenesis imperfecta (OI) and campomelic dysplasia (CD) are the main differential diagnoses of severe HPP, so that in case of negative result for ALPL mutations, OI and CD genes had often to be analyzed, lengthening the time before diagnosis. We report here our 18-month experience in testing 46 patients for HPP and differential diagnosis by targeted NGS and show that this strategy is efficient and useful. We used an array including ALPL gene, genes of differential diagnosis COL1A1 and COL1A2 that represent 90% of OI cases, SOX9, responsible for CD, and 8 potentially modifier genes of HPP. Seventeen patients were found to carry a mutation in one of these genes. Among them, only 10 out of 15 cases referred for HPP carried a mutation in ALPL and 5 carried a mutation in COL1A1 or COL1A2. Interestingly, three of these patients were adults with fractures and/or low BMD. Our results indicate that HPP and OI may be easily misdiagnosed in the prenatal stage but also in adults with mild symptoms for these diseases.