CXCR4 mediates the effects of IGF-1R signaling in rodent bone homeostasis and fracture repair.

Non-union fractures have considerable clinical and economic burdens and yet the underlying pathogenesis remains largely undetermined. The fracture healing process involves cellular differentiation, callus formation and remodeling, and implies the recruitment and differentiation of mesenchymal stem cells that are not fully characterized. C-X-C chemokine receptor 4 (CXCR4) and Insulin-like growth factor 1 receptor (IGF-1R) are expressed in the fracture callus, but their interactions still remain elusive. We hypothesized that the regulation of CXCR4 by IGF-1R signaling is essential to maintain the bone homeostasis and to promote fracture repair. By using a combination of in vivo and in vitro approaches, we found that conditional ablation of IGF-1R in osteochondroprogenitors led to defects in bone formation and mineralization that associated with altered expression of CXCR4 by a discrete population of endosteal cells. These defects were corrected by AMD3100 (a CXCR4 antagonist). Furthermore, we found that the inducible ablation of IGF-1R in osteochondroprogenitors led to fracture healing failure, that associated with an altered expression of CXCR4. In vivo AMD3100 treatment improved fracture healing and normalized CXCR4 expression. Moreover, we determined that these effects were mediated through the IGF-1R/Insulin receptor substrate 1 (IRS-1) signaling pathway. Taken together, our studies identified a novel population of endosteal cells that is functionally regulated through the modulation of CXCR4 by IGF-1R signaling, and such control is essential in bone homeostasis and fracture healing. Knowledge gained from these studies has the potential to accelerate the development of novel therapeutic interventions by targeting CXCR4 signaling to treat non-unions.

The Unraveling: Cardiac and Musculoskeletal Defects and Their Role in Common Alzheimer Disease Morbidity and Mortality.

Alzheimer disease (AD) is characterized by deterioration of cognitive capabilities with an estimated 44 million individuals worldwide living with it. Beyond memory deficits, the most common AD co-morbidities include swallowing defects (muscle), fractures (bone, muscle), and heart failure. The underlying causes of these co-morbidities and their role in AD pathophysiology are currently unknown. This review is the first to summarize the emerging picture of the cardiac and musculoskeletal deficits in human AD. We present the involvement of the heart, characterized by diastolic heart failure, the presence of amyloid deposits, and electrophysiological changes, compared with age-matched control subjects. The characteristic musculoskeletal defects in AD come from recent clinical studies and include potential underlying mechanisms (bone) in animal models. These studies detail a primary muscle weakness (without a loss of muscle mass) in patients with mild cognitive impairment, with progression of cognitive impairment to AD associating with ongoing muscle weakness and the onset of muscle atrophy. We conclude by reviewing the loss of bone density in patients with AD, paralleling the increase in fracture and fall risk in specific populations. These studies paint AD as a systemic disease in broad strokes, which may help elucidate AD pathophysiology and to allow for new ways of thinking about therapeutic interventions, diagnostic biomarkers, and the pathogenesis of this multidisciplinary disease.

Lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence enables long-term secretion of functional chondroitinase ABC in human bone marrow stromal cells.

OBJECTIVE: To test the feasibility of secretion of functional chondroitinase ABC (ChABC), a bacterial enzyme that promotes axonal regeneration after spinal cord injury, from human bone marrow stromal cells (hBMSCs). RESULTS: A lentiviral-expression vector, Lenti6.3-ChABC-3F, carrying the ChABC-3F gene without the bacterial leader sequence (aa 1-24) was constructed. Transfection of these Lenti6.3-ChABC-3F lentivirus led to stable expression in and secrection of ChABC proteins from hBMSCs for at least ten passages in culture in vitro, which was demonstrated by QRT-PCR, immunostaining, western blotting and ELISA. Moreover, the secreted ChABC proteins exhibited similar functional activity as the commercially-available ChABC. CONCLUSIONS: The lentivirus-mediated transfection of chondroitinase ABC gene without the bacterial leader sequence induced substantial long-term secretion of functional ChABC in hBMSCs.

Chondroitin sulfate-E is a negative regulator of a pro-tumorigenic Wnt/beta-catenin-Collagen 1 axis in breast cancer cells.

Expression of the glycosaminoglycan chondroitin sulfate-E (CS-E) is misregulated in many human cancers, including breast cancer. Cell-surface associated CS-E has been shown to have pro-tumorigenic functions, and pharmacological treatment with exogenous CS-E has been proposed to interfere with tumor progression mediated by endogenous CS-E. However, the effects of exogenous CS-E on breast cancer cell behavior, and the molecular mechanisms deployed by CS-E are not well understood. We show here that treatment with CS-E, but not other chondroitin forms, could interfere with the invasive protrusion formation and migration of breast cancer cells in three-dimensional organotypic cultures. Microarray analysis identified transcriptional programs controlled by CS-E in these cells. Importantly, negative regulation of the pro-metastatic extracellular matrix gene Col1a1 was required for the anti-migratory effects of exogenous CS-E. Knock-down of Col1a1 gene expression mimics the effects of CS-E treatment, while exposing cells to a preformed collagen I matrix interfered with the anti-migratory effects of CS-E. In addition, CS-E specifically interfered with Wnt/beta-catenin signaling, a known pro-tumorigenic pathway. Lastly, we demonstrate that Col1a1 is a positively regulated target gene of the Wnt/beta-catenin pathway in breast cancer cells. Together, our data identify treatment with exogenous CS-E as negative regulatory mechanism of breast cancer cell motility through interference with a pro-tumorigenic Wnt/beta-catenin-Collagen I axis.

Biphasic role of chondroitin sulfate in cardiac differentiation of embryonic stem cells through inhibition of Wnt/ß-catenin signaling.

The glycosaminoglycan chondroitin sulfate is a critical component of proteoglycans on the cell surface and in the extracellular matrix. As such, chondroitin sulfate side chains and the sulfation balance of chondroitin play important roles in the control of signaling pathways, and have a functional importance in human disease. In contrast, very little is known about the roles of chondroitin sulfate molecules and sulfation patterns during mammalian development and cell lineage specification. Here, we report a novel biphasic role of chondroitin sulfate in the specification of the cardiac cell lineage during embryonic stem cell differentiation through modulation of Wnt/beta-catenin signaling. Lineage marker analysis demonstrates that enzymatic elimination of endogenous chondroitin sulfates leads to defects specifically in cardiac differentiation. This is accompanied by a reduction in the number of beating cardiac foci. Mechanistically, we show that endogenous chondroitin sulfate controls cardiac differentiation in a temporal biphasic manner through inhibition of the Wnt/beta-catenin pathway, a known regulatory pathway for the cardiac lineage. Treatment with a specific exogenous chondroitin sulfate, CS-E, could mimic these biphasic effects on cardiac differentiation and Wnt/beta-catenin signaling. These results establish chondroitin sulfate and its sulfation balance as important regulators of cardiac cell lineage decisions through control of the Wnt/beta-catenin pathway. Our work suggests that targeting the chondroitin biosynthesis and sulfation machinery is a novel promising avenue in regenerative strategies after heart injury.

Inhibition by chondroitin sulfate E can specify functional Wnt/ß-catenin signaling thresholds in NIH3T3 fibroblasts.

Aberrant activation of the Wnt/ß-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/ß-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/ß-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/ß-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome.

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome. Here we demonstrate a loss of C4S, as well as a reduction in C4ST-1 mRNA and protein expression, in primary fibroblasts from Costello syndrome patients. We go on to show that expression of oncogenic HRAS in normal fibroblasts can repress C4ST-1 expression, whereas interference with oncogenic HRAS signaling in Costello syndrome fibroblasts elevated C4ST-1 expression, thus identifying C4ST-1 as a negatively regulated target gene of HRAS signaling. Importantly, we show that forced expression of C4ST-1 in Costello fibroblasts could rescue the proliferation and elastogenesis defects associated with oncogenic HRAS signaling in these cells. Our results indicate reduced C4ST-1 expression and chondroitin sulfation imbalance mediating the effects of oncogenic HRAS signaling in the pathogenesis of Costello syndrome. Thus, our work identifies C4ST-1-dependent chondroitin sulfation as a downstream vulnerability in oncogenic RAS signaling, which might be pharmacologically exploited in future treatments of not only Costello syndrome and other RASopathies, but also human cancers associated with activating RAS mutations.

Efficient secretion of biologically active Chondroitinase ABC from mammalian cells in the absence of an N-terminal signal peptide.

Proteoglycans carrying chondroitin sulfate side chains have been shown to fulfill important biological functions in development, disease, and signaling. One area of considerable interest is the functional importance of chondroitin sulfates as inhibitors of the regeneration of axonal projections in the mammalian central nervous system. In animal models of spinal cord injury, injections of the enzyme Chondroitinase ABC from the bacterium Proteus vulgaris into the lesion site leads to degradation of chondroitin sulfates, and promotes axonal regeneration and significant functional recovery. Here, a mammalian expression system of an epitope-tagged Chondroitinase ABC protein is described. It is demonstrated that the addition of a eukaryotic secretion signal sequence to the N-terminus of the bacterial Chondroitinase ABC sequence allowed secretion, but interfered with function of the secreted enzyme. In contrast, expression of the Chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence led to efficient secretion of a biologically active Chondroitinase ABC protein from both immortalized and primary cells. Moreover, the C-terminal epitope tag could be utilized to follow expression of this protein. This novel Chondroitinase ABC gene is a valuable tool for a better understanding of the in vivo roles of chondroitin sulfates in mammalian development and disease, as well as in gene therapy approaches, including the treatment of spinal chord injuries.

The roles of chondroitin-4-sulfotransferase-1 in development and disease.

The glycosaminoglycan chondroitin sulfate (CS) consists of long linear chains of repeating disaccharide units, which are covalently attached to core proteins to form CS-proteoglycans. These molecules have been shown to fulfill important biological functions in development, disease, and signaling. Biosynthesis of CS takes place in the Golgi apparatus. Concomitant to chondroitin chain elongation, sulfation of specific carbon residues by chondroitin sulfotransferase enzymes takes place. The sulfation balance and pattern of CS on specific carbon residues are tightly regulated during development, injury, and disease, with the temporal and spatial expression of chondroitin sulfotransferase genes believed to be a crucial determinant of this fine balance of chondroitin sulfation. Chondroitin-4-sulfotransferase-1 (C4ST-1)/carbohydrate sulfotransferase 11 (CHST11) is one of the enzymes involved in the sulfation of chondroitin by catalyzing the transfer of sulfate groups from a sulfate donor to the carbon-4 position of the N-acetylgalactosamine sugar of the repeating disaccharide units. Here, I summarize the significant recent advances in our understanding of the roles of C4ST-1 in vertebrate development, disease, and signaling pathways, and the transcriptional regulation of the C4ST-1 gene. Proper 4-sulfation of chondroitin by C4ST-1 plays a crucial role in the skeletal development and signaling events, and new evidence is suggestive of a potential role for C4ST-1 in human disease, including cancer.

Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis.

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate are polysaccharide chains that are attached to core proteins to form proteoglycans. The biosynthesis of GAGs is a multistep process that includes the attachment of sulfate groups to specific positions of the polysaccharide chains by sulfotransferases. Heparan-sulfate and heparan sulfate-sulfotransferases play important roles in growth factor signaling and animal development. However, the biological importance of chondroitin sulfation during mammalian development and growth factor signaling is poorly understood. We show that a gene trap mutation in the BMP-induced chondroitin-4-sulfotransferase 1 (C4st1) gene (also called carbohydrate sulfotransferase 11 - Chst11), which encodes an enzyme specific for the transfer of sulfate groups to the 4-O-position in chondroitin, causes severe chondrodysplasia characterized by a disorganized cartilage growth plate as well as specific alterations in the orientation of chondrocyte columns. This phenotype is associated with a chondroitin sulfation imbalance, mislocalization of chondroitin sulfate in the growth plate and an imbalance of apoptotic signals. Analysis of several growth factor signaling pathways that are important in cartilage growth plate development showed that the C4st1(gt/gt) mutation led to strong upregulation of TGFbeta signaling with concomitant downregulation of BMP signaling, while Indian hedgehog (Ihh) signaling was unaffected. These results show that chondroitin 4-O-sulfation by C4st1 is required for proper chondroitin sulfate localization, modulation of distinct signaling pathways and cartilage growth plate morphogenesis. Our study demonstrates an important biological role of differential chondroitin sulfation in mammalian development.

Turning it up a Notch: cross-talk between TGF beta and Notch signaling.

Signaling through both the transforming growth factor beta (TGF beta) superfamily of growth factors and Notch play crucial roles during embryonic pattern formation and cell fate determination. Although both pathways are able to exert similar biological responses in certain cell types, a functional interaction between these two signaling pathways has not been described. Now, three papers provide evidence of both synergy and antagonism between TGF beta and Notch signaling. These reports describe a requirement for Notch signal transducers in TGF beta- and BMP-induced expression of Notch target genes, as well as in BMP-controlled cell differentiation and migration. These papers uncover a direct link between the Notch and TGF beta pathways and suggest a critical role for Notch in some of the biological responses to TGF beta family signaling.

Myocardial storage of chondroitin sulfate-containing moieties in Costello syndrome patients with severe hypertrophic cardiomyopathy.

Costello syndrome is a distinctive multiple congenital anomaly syndrome, characterized by loose soft skin with deep palmar and plantar creases, loose joints, distinctive coarse facial features, skeletal abnormalities, cardiac abnormalities (cardiovascular malformation (CVM), hypertrophic cardiomyopathy, tachycardia), predisposition to malignancy, developmental delays, and mental retardation. Previous studies with cultured fibroblasts from individuals with Costello syndrome demonstrate excessive accumulation of chondroitin sulfate-bearing proteoglycans, associated with both impaired formation of elastic fibers and an unusually high rate of cellular proliferation. Despite multiple clinical reports of cardiac abnormalities, there has been only one previously published report describing post-mortem findings in hearts from Costello syndrome patients. Here we provide a detailed description of the post-mortem findings of the hearts of three children with Costello syndrome. Routine histological examination and results of targeted histochemical and immunohistochemical studies revealed that in addition to cardiomyocyte hypertrophy, these hearts also demonstrated massive pericellular and intracellular accumulation of chondroitin sulfate-bearing proteoglycans and a marked reduction of elastic fibers. Normal stroma was replaced by multifocal collagenous fibrosis. Most peculiar was the finding that the bulk of the chondroitin sulfate accumulated in these Costello syndrome hearts is a chondroitin-6-sulfate. In contrast, deposition of chondroitin-4 sulfate was below the level detected in normal hearts. We propose that an imbalance in sulfation of chondroitin sulfate molecules and subsequent accumulation of chondroitin-6-sulfate in cardiomyocytes contribute to the development of the hypertrophic cardiomyopathy of Costello syndrome.

Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages.

Loss-of-function mutations in the murine dominant white spotting/c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.

Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis.

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor beta (TGFbeta) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.

A high-throughput induction gene trap approach defines C4ST as a target of BMP signaling.

Here we describe a novel gene trap protocol to screen for target genes that are regulated during inductive events in undifferentiated and differentiated mouse embryonic stem cells. This approach integrates several features that allows in vitro screening of large numbers of gene trap clones prior to generating lines of mutant mice. Moreover, targets of spatially and temporally restricted signaling pathways can be analyzed by screening undifferentiated ES cells versus ES cells differentiated into embryoid bodies. We employed this protocol to screen 1920 gene trap lines to identify targets and mediators of signaling through three growth factors of the TGFbeta superfamily--BMP2, activin and nodal. We identified two genes that are induced by BMP2 in a differentiation-dependent manner. One of the genes encodes for Chondroitin-4-sulfotransferase and displays a highly specific temporal and spatial expression pattern during mouse embryogenesis. These results demonstrate the feasibility of a high-throughput gene trap approach as a means to identify mediators and targets of multiple growth factor signaling pathways that function during different stages of development.