Two-day-treatment of Activin-A leads to transient change in SV-HFO osteoblast gene expression and reduction in matrix mineralization.

Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin-A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression profile. A single 2-day treatment of Activin-A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A-treated osteoblasts responded with transient change in gene expression, in a 2-wave-fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1-2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin-A. In summary, 2-day treatment with Activin-A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes.

The microtubule plus-end tracking protein CLASP2 is required for hematopoiesis and hematopoietic stem cell maintenance.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression.

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

GPM6B regulates osteoblast function and induction of mineralization by controlling cytoskeleton and matrix vesicle release.

Neuronal membrane glycoprotein gene (GPM6B) encodes a membrane glycoprotein that belongs to the proteolipid protein family. We identified GPM6B as a gene that is strongly upregulated during osteoblast differentiation. To investigate the role of GPM6B in the process of bone formation, we silenced GPM6B expression during osteogenic differentiation of human mesenchymal stem cells (hMSCs). GPM6B silencing in hMSCs resulted in reduced alkaline phosphate (ALP) activity along with reduced mineralization of extracellular matrix (ECM). Microarray expression analysis of GPM6B-depleted osteogenic hMSCs revealed significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry results confirm changes in the distribution of actin filaments, as well as the shape and size of focal adhesions on GPM6B silencing. Moreover, we demonstrated that production and release of ALP-positive matrix vesicles (MVs) were reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast function and bone formation. This finding demonstrates the significance of cytoskeleton organization for MV production and subsequent mineralization.

A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone.

Bone-marrow adipogenesis is an aging-related phenomenon and is correlated with osteoporosis. The latter is a prevalent bone disease in the elderly leading to increased fracture risk and mortality. It is widely hypothesized that the underlying molecular mechanism includes a shift in the commitment of mesenchymal stem cells (MSCs) from the osteogenic lineage to the adipogenic lineage. Lineage skewing is at least partially a result of transcriptional changes. The nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPAR-gamma) has been proposed as a major decision factor in MSC lineage commitment, promoting adipogenesis at the expense of osteogenesis. Here we found that PPAR-gamma acted unexpectedly to stimulate osteoblast differentiation from human bone marrow-derived MSCs. Both rosiglitazone-mediated activation and overexpression of PPAR-gamma caused acceleration of osteoblast differentiation. Conversely, shRNAi-mediated PPAR-gamma knockdown diminished osteoblast differentiation. MSCs that were treated with rosiglitazone did not preferentially differentiate into adipocytes. However, the rosiglitazone-mediated acceleration of osteoblast differentiation was followed by increased accumulation of reactive oxygen species and apoptosis. In contrast to the osteogenic lineage, cells of the adipogenic lineage were protected from this. Our data support a new concept on bone health that adds to the explanation of the clinically observed suppressive action of activated PPAR-gamma on bone and the associated phenomenon of bone marrow adipogenesis. This concept is based on a higher susceptibility of the osteogenic than the adipogenic lineage to oxidative stress and apoptosis that is preferentially triggered in the osteoblasts by activated PPAR-gamma.

Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules.

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (+/- 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (+/- 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states.

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility.

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.

The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis.

CLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP-170 is detected in lungs and embryos of homozygous CLIP-170 knockout mice, but not in other tissues and cell types, indicating that we have generated a hypomorphic mutant. Homozygous CLIP-170 knockout mice are viable and appear normal. However, male knockout mice are subfertile and produce sperm with abnormal heads. Using the knock-in mice, we followed GFP-CLIP-170 expression and behavior in dissected, live testis tubules. We detect plus-end-tracking GFP-CLIP-170 in spermatogonia. As spermatogenesis proceeds, GFP-CLIP-170 expression increases and the fusion protein strongly marks syncytia of differentiated spermatogonia and early prophase spermatocytes. Subsequently GFP-CLIP-170 levels drop, but during spermiogenesis (post-meiotic development), GFP-CLIP-170 accumulates again and is present on spermatid manchettes and centrosomes. Bleaching studies show that, as spermatogenesis progresses, GFP-CLIP-170 converts from a mobile plus-end-tracking protein to a relatively immobile protein. We propose that CLIP-170 has a structural function in the male germline, in particular in spermatid differentiation and sperm head shaping.

Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes.

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 microm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3-green fluorescent protein (GFP)-expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 microm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 microm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100-permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 microm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactin-dynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation.