Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Gain of glycosylation: a new pathomechanism of myelin protein zero mutations.

We report the first case of a missense mutation in MPZ causing a gain of glycosylation in myelin protein zero, the main protein of peripheral nervous system myelin. The patient was affected by a severe demyelinating neuropathy caused by a missense mutation, D32N, that created a new glycosylation sequence. We confirmed that the mutant protein is hyperglycosylated, is partially retained into the Golgi apparatus in vitro, and disrupts intercellular adhesion. By sequential experiments, we demonstrated that hyperglycosylation is the main mechanism of this mutation. Gain of glycosylation is a new mechanism in Charcot-Marie-Tooth type 1B.

TTF-1/NKX2.1 up-regulates the in vivo transcription of nestin.

TTF-1/NKX2.1, also known as T/EBP, is a homeodomain-containing gene involved in the organogenesis of the thyroid gland, lung and ventral forebrain. We have already reported that in 3T3 cells, TTF-1/NKX2.1 up-regulates the transcription of nestin, an intermediate filament protein expressed in multipotent neuroepithelial cells, by direct DNA-binding to a HRE/CRE-like site (NestBS) within a CNS-specific enhancer. Here, we demonstrate that TTF-1/NKX2.1 is co-expressed with nestin in the embryonal forebrain. We also performed a transgenic mouse embryo analysis in which NestBS was replaced by the canonical TTF-1/NKX2.1 consensus DNA-binding site (as identified in many thyroid- and lung-specific genes and very divergent from NestBS) or a random mutation. We observed beta-galactosidase expression in forebrain regions where TTF-1/NKX2.1 is expressed in wild-type embryos, and -to a minor extent- in rostralmost telencephalic regions and thalamus, whereas no beta-galactosidase expression was detected in forebrains of embryos bearing the random mutation. These data show that TTF-1/NKX2.1 regulates the transcription of the nestin gene in vivo through the NestBS site, suggesting that nestin might be at least one of the effectors of TTF-1/NKX2.1 during forebrain development. Finally, we have shown that the transactivating effect of TTF-1/NKX2.1 on the CNS-specific enhancer is unaffected by Retinoic Acid Receptor-alpha.

Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains.

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrin-coated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization.