Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Structural determinants involved in the regulation of CXCL14/BRAK expression by the 26 S proteasome

The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells; however, in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation.     

Erratum to: Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell transplants

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.     

The chemokine BRAK/CXCL14 regulates synaptic transmission in the adult mouse dentate gyrus stem cell niche

The chemokine BRAK/CXCL14 is an ancient member of the chemokine family whose functions in the brain are completely unknown. We examined the distribution of CXCL14 in the nervous system during development and in the adult. Generally speaking, CXCL14 was not expressed in the nervous system prior to birth, but it was expressed in the developing whisker follicles (E14.5) and subsequently in the hair follicles and skin. Postnatally, CXCL14 was also highly expressed in many regions of the brain, including the cortex, basal ganglia, septum and hippocampus. CXCL14 was also highly expressed in the dorsal root ganglia. We observed that in the hippocampal dentate gyrus (DG) CXCL14 was expressed by GABAergic interneurons. We demonstrated that CXCL14 inhibited GABAergic transmission to nestin-EGFP-expressing neural stem/progenitor cells in the adult DG. CXCL14 inhibited both the tonic and phasic effects of synaptically released GABA. In contrast CXCL12 enhanced the effects of GABA at these same synapses. CXCL14 increased [Ca(2+)](i) in neural stem cells cultured from the postnatal brain indicating that they expressed the CXCL14 receptor. These observations are consistent with the view that CXCL12 and CXCL14 may normally act as positive and negative regulators of the effects of GABA in the adult DG stem cell niche.     

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells

Abstract

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and hydroxyl radical (HO^?), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO^? than with H₂O₂. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H₂O₂ or HO^? stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.     

P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling

P-cadherin belongs to the family of Ca²⁺-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.     

Altered T cell differentiation and Notch signaling induced by the ectopic expression of keratin K10 in the epithelial cells of the thymus

Transgenic mice expressing hK10 under the keratin K5 promoter display several alterations in the epidermis including decreased cell proliferation, and reduced susceptibility to tumor development. Given that K5 promoter is also active in the epithelial cells of the thymus, we explored the possible alterations of the thymus because of K10 transgene expression. We found severe thymic alterations, which affect not only the thymic epithelial cells (TEC), but also thymocytes. We observed altered architecture and premature thymus involution in the transgenic mice associated with increased apoptosis and reduced proliferation of the thymocytes. Interestingly, prior to the development of this detrimental phenotype, thymocytes of the transgenic mice also displayed altered differentiation, which is aggravated later on. Molecular characterization of this phenotype indicated that Akt activity is reduced in TEC, but not in thymocytes. In addition, we also observed altered expression of Notch family members and some of their ligands both in TEC and T cells. This produces reduced Notch activity in TEC but increased Notch activity in thymocytes, which is detectable prior to the disruption of the thymic architecture. In addition, we also detect altered Notch expression in the epidermis of bK5hK10 transgenic mice. Collectively the present data indicate that keratin K10 may induce severe alterations not only in a cell autonomous manner, but also in neighboring cells by the modulation of signals involved in cell-cell interactions.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.     

Connexin 43 contributes to differentiation of retinal pigment epithelial cells via cyclic AMP signaling

Retinal pigment epithelium (RPE) cells play important roles in the visual system that supports neurosensory retina homeostasis. Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJIC) participates in the regulation of retinal organogenesis, but much of the function of Cx43 on the differentiation of RPE cells is unclear. Here, we report the involvement of Cx43 in RPE differentiation. Knockdown of Cx43 in RPE cells dramatically inhibited the differentiation, whereas Cx43-overexpression successfully induced RPE cell differentiation under de-differentiation conditions. From the experiments using GJIC inhibitors and C-terminus-truncated mutant of Cx43, it was clearly demonstrated that the regulation of RPE cell differentiation by Cx43 did not result from Cx43-mediated GJIC. The RPE cell differentiation induced by Cx43-overexpression was abolished by a cAMP antagonist. In contrast, the treatment with forskolin and phosphodiesterase inhibitor rolipram induced RPE cell differentiation under de-differentiation conditions. These findings indicate that Cx43 contributes to RPE differentiation via cAMP signaling.