Project description:Indian hedgehog (Ihh) regulates proliferation and differentiation of chondrocytes in the growth plate. Although the biology of Ihh is currently well documented, its transcriptional regulation is poorly understood. delta-EF1 is a two-handed zinc finger/homeodomain transcriptional repressor. Targeted inactivation of mouse delta-EF1 leads to skeletal abnormalities including disorganized growth plates, shortening of long bones, and joint fusions, which are reminiscent of defects associated with deregulation of Ihh signaling. Here, we show that the absence of delta-EF1 results in delayed hypertrophic differentiation of chondrocytes and increased cell proliferation in the growth plate. Further, we demonstrate that delta-EF1 binds to the putative regulatory elements in intron 1 of Ihh in vitro and in vivo, resulting in down-regulation of Ihh expression. Finally, we show that delta-EF1 haploinsufficiency leads to a postnatal increase in trabecular bone mass associated with enhanced Ihh expression. In summary, we have identified delta-EF1 as an in vivo negative regulator of Ihh expression in the growth plate.
Project description:Microtubules are noncovalent polymers built from αβ-tubulin dimers. The disordered C-terminal tubulin tails are functionalized with multiple glutamate chains of variable lengths added and removed by tubulin tyrosine ligases (TTLLs) and carboxypeptidases (CCPs). Glutamylation is abundant on stable microtubule arrays such as in axonemes and axons, and its dysregulation leads to human pathologies. Despite this, the effects of glutamylation on intrinsic microtubule dynamics are unclear. Here we generate tubulin with short and long glutamate chains and show that glutamylation slows the rate of microtubule growth and increases catastrophes as a function of glutamylation levels. This implies that the higher stability of glutamylated microtubules in cells is due to effectors. Interestingly, EB1 is minimally affected by glutamylation and thus can report on the growth rates of both unmodified and glutamylated microtubules. Finally, we show that glutamate removal by CCP1 and 5 is synergistic and occurs preferentially on soluble tubulin, unlike TTLL enzymes that prefer microtubules. This substrate preference establishes an asymmetry whereby once the microtubule depolymerizes, the released tubulin is reset to a less-modified state, while polymerized tubulin accumulates the glutamylation mark. Our work shows that a modification on the disordered tubulin tails can directly affect microtubule dynamics and furthers our understanding of the mechanistic underpinnings of the tubulin code.
Project description:Merlin is encoded by the neurofibromatosis type 2 (NF2) gene and is a member of the Band 4.1 protein family. This protein acts as a linker that connects cell surface proteins to the actin cytoskeleton. Defects caused by mutations of the NF2 gene give rise to NF2 disease, which is generally characterized by the formation of bilateral vestibular schwannomas and, to a lesser extent, meningiomas and ependymomas. In addition to these tumor types, NF2 is mutated and/or merlin expression is reduced or lost in numerous non-NF2 associated tumors, including melanoma. However, the role of merlin in human melanoma growth and the mechanism underlying its effect are currently unknown. In the present study, we show that merlin knockdown enhances melanoma cell proliferation, migration, and invasion in vitro and that decreased merlin expression promotes subcutaneous melanoma growth in immunocompromised mice. Concordantly, we find that increased expression of merlin in a metastatic melanoma cell line reduced their in vitro migration and proliferation, and diminished their ability to grow in an anchorage independent manner. Increased merlin expression also inhibits in vivo growth of these melanoma cells. Lastly, we demonstrate that higher merlin levels in human melanoma cells promote the H(2)O(2)-induced activation of MST1/2 Ser/Thr kinases, which are known tumor suppressors in the Hippo signaling pathway. Taken together, these results provide for the first time evidence that merlin negatively regulates human melanoma growth, and that loss of merlin, or impaired merlin function, results in an opposite effect. In addition, we show that increased merlin expression leads to enhanced activation of the MTS1/2 kinases, implying the potential roles of MST1/2 in mediating the anti-melanoma effects of merlin.
Project description:Microtubule dynamics are dominated by events at microtubule plus ends as they switch between discrete phases of growth and shrinkage. Through their ability to generate force and direct polar cell transport, microtubules help to organise global cell shape and polarity. Conversely, because plus-end binding proteins render the dynamic instability of individual microtubules sensitive to the local intracellular environment, cyto-architecture also affects the overall distribution of microtubules. Despite the importance of plus-end regulation for understanding microtubule cytoskeletal organisation and dynamics, little is known about the signalling mechanisms that trigger changes in their behaviour in space and time. Here, we identify a microtubule-associated kinase, Drosophila Tao-1, as an important regulator of microtubule stability, plus-end dynamics and cell shape. Active Tao-1 kinase leads to the destabilisation of microtubules. Conversely, when Tao-1 function is compromised, rates of cortical-induced microtubule catastrophe are reduced and microtubules contacting the actin cortex continue to elongate, leading to the formation of long microtubule-based protrusions. These data reveal a role for Tao-1 in controlling the dynamic interplay between microtubule plus ends and the actin cortex in the regulation of cell form.
Project description:Transforming growth factor (TGF)-? signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-? signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-? signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-? signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-? type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-?-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-?. Consistently, TGF-?-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-? signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-? signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-? signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-? signaling in a coordinated manner when cells are stimulated with high levels of TGF-?.
Project description:ETS is a family of transcriptional regulators with functions in most biological processes. Dysregulated ETS factor function leads to altered expression of multiple genes that play critical roles in many of the processes required for cancer progression. While the Ets family gene, prostate-derived ETS factor (PDEF), is expressed in epithelial tissues including prostate, breast, and colon, PDEF protein expression has been found to be reduced or lost during prostate and breast cancer progression. The goal of this study was to examine the expression and biologic impact of altered PDEF expression in colon cancer. PDEF mRNA and protein are not detectable in several colon-cancer-derived cell lines. Re-expression of PDEF in colon cancer cells inhibits growth and migration. Growth affects are due to altered cellular proliferation, indicated by increased altered cell population in G(1) and S phases of the cell cycle, as well as increased apoptosis. Relevant to its modulation of growth and migration phenotypes, PDEF expression resulted in altered expression of genes with established roles in cell cycle, motility, and invasion. Furthermore, chromatin immunoprecipitation studies show that p21 and urokinase plasminogen activator (uPA) are direct PDEF transcriptional targets. While non-tumor colon epithelium expresses PDEF mRNA and protein, the majority of tumors showed decreased mRNA and/or protein expression. In human tumor tissue samples, PDEF expression was inversely correlated with the expression levels of uPA. Collectively, the data support the model that PDEF is a negative regulator of tumor progression by modulating the expression of growth and migration promoting genes.
Project description:A recent analysis of the hexokinase (HXK) gene family from Arabidopsis revealed that three hexokinase-like (HKL) proteins lack catalytic activity, but share about 50% identity with the primary glucose (glc) sensor/transducer protein AtHXK1. Since the AtHKL1 protein is predicted to bind glc, although with a relatively decreased affinity, a reverse genetics approach was used to test whether HKL1 might have a related regulatory function in plant growth. By comparing phenotypes of an HKL1 mutant (hkl1-1), an HXK1 mutant (gin2-1), and transgenic lines that overexpress HKL1 in either wild-type or gin2-1 genetic backgrounds, it is shown that HKL1 is a negative effector of plant growth. Interestingly, phenotypes of HKL1 overexpression lines are generally very similar to those of gin2-1. These are quantified, in part, as reduced seedling sensitivity to high glc concentrations and reduced seedling sensitivity to auxin-induced lateral root formation. However, commonly recognized targets of glc signalling are not apparently altered in any of the HKL1 mutant or transgenic lines. In fact, most, but not all, of the observed phenotypes associated with HKL1 overexpression occur independently of the presence of HXK1 protein. The data indicate that HKL1 mediates cross-talk between glc and other plant hormone response pathways. It is also considered Whether a possibly decreased glc binding affinity of HKL1 could possibly be a feedback mechanism to limit plant growth in the presence of excessive carbohydrate availability is further considered.
Project description:Dendrite size and morphology are key determinants of the functional properties of neurons and neural circuits. Here we show that CD40, a member of the TNF receptor superfamily, is a major regulator of dendrite growth and elaboration in the developing brain. The dendrites of hippocampal excitatory neurons were markedly stunted in Cd40-/- mice, whereas those of striatal inhibitory neurons were much more exuberant. These striking and opposite phenotypic changes were also observed in excitatory and inhibitory neurons cultured from Cd40-/- mice and were rescued by soluble CD40. The changes in excitatory and inhibitory neurons cultured from Cd40-/- mice were mimicked in neurons of Cd40+/+ mice by treatment with soluble CD40L and were dependent on PKC-β and PKC-γ, respectively. These results suggest that CD40-activated CD40L reverse signalling has striking and opposite effects on the growth and elaboration of dendrites among major classes of brain neurons by PKC-dependent mechanisms.
Project description:BACKGROUND:Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ? (TGF-?) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-?1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS:We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS:We demonstrate that LMO7 is induced postinjury and by TGF-? in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-? signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-? treatment. TGF-? neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-? signaling by inducing expression of Tgfb1 mRNA, TGF-? protein, ?v and ?3 integrins that promote activation of latent TGF-?, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-? autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS:LMO7 is induced by TGF-? and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-? pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.
Project description:Neural connectivity requires neuronal differentiation, axon growth, and precise target innervation. Midbrain dopaminergic neurons project via the nigrostriatal pathway to the striatum to regulate voluntary movement. While the specification and differentiation of these neurons have been extensively studied, the molecular mechanisms that regulate midbrain dopaminergic axon growth and target innervation are less clear. Here we show that the transcription factor Zeb2 cell-autonomously represses Smad signalling to limit midbrain dopaminergic axon growth and target innervation. Zeb2 levels are downregulated in the embryonic rodent midbrain during the period of dopaminergic axon growth, when BMP pathway components are upregulated. Experimental knockdown of Zeb2 leads to an increase in BMP-Smad-dependent axon growth. Consequently there is dopaminergic hyperinnervation of the striatum, without an increase in the numbers of midbrain dopaminergic neurons, in conditional Zeb2 (Nestin-Cre based) knockout mice. Therefore, these findings reveal a new mechanism for the regulation of midbrain dopaminergic axon growth during central nervous system development.