RESUMEN

The transcription factor Sox11 is expressed at high levels in developing sensory neurons and injured adult neurons but little is known about its transcriptional targets and function. In this study we examined the role of Sox11 using Neuro2a neuroblastoma cells and cultured mouse dorsal root ganglia (DRG) neurons. Results show Sox11 has an essential role in regulation of neuron survival and neurite outgrowth in Neuro2a cells and primary sensory neurons. Neuro2a cells increase expression of Sox11 as they differentiate in culture. Following addition of 20 microM retinoic acid (RA), a stimulus for differentiation that enhances neurite growth and differentiation, Sox11 level rises. RNAi-mediated knockdown of Sox11 in RA-differentiated Neuro2a cells caused a decrease in neurite growth and an increase in the percent of apoptotic cells. RNA expression analysis showed that Sox11 knockdown modulated the level of mRNAs encoding several genes related to cell survival and death. Further validation in the Neuro2a model showed Sox11 knockdown increased expression of the pro-apoptotic gene BNIP3 (BclII interacting protein 1 NIP3) and decreased expression of the anti-apoptotic gene TANK (TNF receptor-associated factor family member-associated NFkappaB activator). Cultured primary DRG neurons also express Sox11 and treatment with Sox11 small interfering RNA (siRNA) caused a significant decrease in neurite growth and branching and a decrease in mRNA encoding actin-related protein complex 3 (Arpc3), an actin organizing protein that may be involved in axon growth. The percent of apoptotic neurons also increased in cultures of DRG neurons treated with Sox11 siRNA. Similar to Neuro2a cells, a decrease in TANK gene expression occurred, suggesting at least some overlap in Sox11 transcriptional targets in Neuro2a and DRG neurons. These data are consistent with a central role for Sox11 in regulating events that promote neurite growth and neuron survival.

Asunto(s)

Proteínas del Grupo de Alta Movilidad/fisiología , Neuritas/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Recuento de Células/métodos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Interacciones Farmacológicas , Ganglios Espinales/citología , Regulación de la Expresión Génica/genética , Proteínas del Grupo de Alta Movilidad/genética , Inmunohistoquímica/métodos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neuritas/efectos de los fármacos , Neuroblastoma , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción SOXC , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patología , Factores de Tiempo , Transfección/métodos , Tretinoina/farmacología

RESUMEN

The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence. The expression of each of the three corneal KSPGs in the developing and mature chick cornea was investigated by competitive PCR and immuno-biochemical analysis of corneal extracts. Competitive PCR was used to determine the message levels for chick lumican, keratocan and osteoglycin in embryonic day 9, 12, 15, 18 and adult corneas. Results showed that lumican mRNA fluctuated during development but remained at a relatively high level while keratocan and osteoglycin message levels declined steadily from day 9 to adult. Additionally, lumican mRNA was present at higher levels, during all stages of corneal development, than keratocan and at much higher levels than osteoglycin. Antibodies shown to be specific for each KSPG were used to characterize proteoglycans isolated from embryonic and adult chick corneas. KSPGs from embryonic corneas eluted 1-2 fractions earlier on Q-Sepharose than KSPG from adult corneas. Additionally, Western blot analysis showed that embryonic KSPGs were more keratanase-resistant, endo-beta-galactosidase sensitive than adult KSPGs. The results of this study indicate an alteration in sulfation or the fine structure of the glycosaminoglycan chains occurs during corneal maturation for the 3 KSPGs.

Asunto(s)

Pollos/crecimiento & desarrollo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/crecimiento & desarrollo , Sulfato de Queratano/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Embrión de Pollo , Pollos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/genética , Córnea/metabolismo , ADN Complementario/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Sulfato de Queratano/genética , Lumican , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteoglicanos/metabolismo

RESUMEN

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.

Asunto(s)

Sulfatos de Condroitina/metabolismo , Proteínas de la Matriz Extracelular , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/metabolismo , Procesamiento Proteico-Postraduccional , Proteoglicanos/metabolismo , Agrecanos , Animales , Western Blotting , Células COS , Glicosilación , Lectinas Tipo C , Ratones , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

A human corneal fibroblast cDNA library was screened with a bovine lumican cDNA probe to obtain three clones. Sequencing of the longest clone (1.75 kb) yielded an open reading frame of 1014 bp coding for a 338-amino-acid core protein. Amino acid sequencing of a tryptic peptide resulted in a 9-amino-acid match with the derived primary structure, confirming the identity of these clones. Human lumican displays all of the features of small interstitial proteoglycans: N- and C-terminal domains with highly conserved cysteines and a central domain containing nine repeats of slight variations of the leucine motif LXXLXLXXNXL. Like bovine lumican, the human core protein contains four possible N-glycosylation sites in the central domains, all or some of which are substituted with keratan sulfate side chains. At the amino acid level, it is 90% identical with bovine and 72% identical with the chicken core protein. The gene (LUM) was localized to human chromosome 12 by hybridizing a cDNA probe to a Southern blot containing a human/hamster monochromosomal mapping panel DNA. Further sublocalization to 12q21.3-q22 was performed by the fluorescence in situ hybridization technique using a lumican P1 genomic clone. By immunohistochemical staining, we show lumican's presence, not only in the corneal stroma as shown previously, but also in the dermal area of the skin, indicating a wider distribution of this proteoglycan.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/genética , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Sulfato de Queratano/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/metabolismo , Cricetinae , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Sulfato de Queratano/metabolismo , Lumican , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Piel/metabolismo , Especificidad de la Especie

RESUMEN

PURPOSE: To evaluate the expression of lumican and decorin, the major proteoglycans of the adult corneal stroma, during the acquisition of corneal transparency in developing chick embryos. METHODS: mRNA levels of decorin and lumican were measured in total RNA extracted from corneas of days 9 to 18 of development by Northern blot analysis using a 32P-labeled cDNA clone to each proteoglycan. The synthesis lumican and decorin precursor proteins were determined by biosynthetically radiolabeling corneas from day 7 to 18 chick embryos with 35S-methionine, and then using antibodies specific for lumican and decorin core proteins to precipitate the radiolabeled precursor proteins. The accumulation of lumican and decorin was determined by fractionating extracts of day 7 to 18 embryonic corneas by DEAE chromatography into glycoprotein and proteoglycan fractions, and then analyzing each fraction by Western blot using antibodies to lumican and decorin. RESULTS: Lumican and decorin mRNA increased from day 9 to day 18, with respect to beta-actin. The rate of decorin precursor protein synthesis remained relatively low and constant throughout development, but lumican precursor protein synthesis increased dramatically between days 7 and 9 of embryonic development, to a value 80-fold higher than that of decorin, and then decreased exponentially through day 18. Lumican with polylactosamine (nonsulfated keratan sulfate) side chains was detected in extracts of corneas as early as day 7 of embryonic development, and continued to accumulate within the cornea through day 18. Decorin and lumican with sulfated glycosaminoglycan side chains (ie, proteoglycans), however, were not detected in corneal extracts until day 15, when transparency starts to increase, and then accumulated considerably within the cornea by day 18. CONCLUSIONS: The results of these studies suggest that decorin and lumican expression are independently regulated during the period of acquisition of corneal transparency. The switch in production of the polylactosamine form of lumican to the proteoglycan form of lumican at the onset of increasing corneal transparency suggests that the sulfation of lumican may be important for the development of corneal transparency.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/embriología , Córnea/metabolismo , Sulfato de Queratano/metabolismo , Polimorfismo Genético , Actinas/genética , Actinas/metabolismo , Amino Azúcares/biosíntesis , Amino Azúcares/aislamiento & purificación , Animales , Northern Blotting , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Cromatografía por Intercambio Iónico , Sondas de ADN , Decorina , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular , Expresión Génica , Sulfato de Queratano/genética , Sulfato de Queratano/aislamiento & purificación , Lumican , Polisacáridos/biosíntesis , Polisacáridos/aislamiento & purificación , Proteoglicanos/genética , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo

RESUMEN

Corneal transparency is dependent on the size and arrangement of collagen fibrils within the corneal stroma. The corneal stroma is composed primarily of collagen type 1 fibrils and two proteoglycans: one with chondroitin/dermatan sulfate side-chains (decorin) and one with keratan sulfate side-chains (lumican). We investigated the effects of the corneal proteoglycans on corneal collagen fibrillogenesis, utilizing an in vitro assay for fibril formation. Collagen was extracted from bovine corneal stromas with 0.1 M acetic acid and monomers purified by NaCl precipitation. Decorin and lumican were extracted from bovine corneal stroma with either 0.7 M NaCl or 4 M guanidine HCl and purified by DEAE and Sepharose CL-4B chromatography. Decorin and lumican from both extracts inhibited the rate of collagen fibrillogenesis and the development of turbidity in fibrillogenesis samples. Furthermore, the core proteins of decorin and lumican were shown to be as effective as the intact proteoglycans in inhibiting fibrillogenesis. The decorin core protein isolated from the 0.7 M NaCl extract was determined to be a 20 kDa fragment which lacks the C-terminal half of the core protein. This fragment was approximately 1/36 as effective in inhibiting fibrillogenesis as intact decorin isolated from guanidine extracts. This suggests that the C-terminal half of the decorin core plays an important role in the interaction of this proteoglycan with collagen. Lumican extracted with 0.7 M NaCl was slightly smaller and was only one-sixth as effective in inhibiting collagen fibril formation as 4 M guanidine extracted lumican. Furthermore reduction and alkylation of lumican core protein abolished the inhibitory activity of the core protein on collagen fibrillogenesis. Electron microscopic examination indicated that fibrils formed in the presence of lumican and lumican core protein were significantly thinner than fibrils formed in the absence of proteoglycans. The results of these studies indicate that in addition to decorin, lumican retards corneal collagen fibrillogenesis and results in the formation of collagen fibrils which are significantly thinner than those formed in the absence of any proteoglycan. The inhibitory activity of lumican or decorin on collagen fibrillogenesis resides in he core proteins of these proteoglycans, not the glycosaminoglycan side chains, and that interaction of the lumican core protein with collagen appears to be dependent on the presence of disulfide bridges within the protein core.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/farmacología , Colágeno/metabolismo , Sustancia Propia/metabolismo , Sulfato de Queratano/farmacología , Proteoglicanos/farmacología , Alquilación , Secuencia de Aminoácidos , Animales , Bovinos , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Colágeno/aislamiento & purificación , Colágeno/ultraestructura , Sustancia Propia/efectos de los fármacos , Sustancia Propia/ultraestructura , Decorina , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular , Sulfato de Queratano/aislamiento & purificación , Lumican , Microscopía Electrónica , Datos de Secuencia Molecular , Oxidación-Reducción , Proteoglicanos/aislamiento & purificación

RESUMEN

The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Córnea/química , Sulfato de Queratano/aislamiento & purificación , Proteoglicanos/aislamiento & purificación , Animales , Western Blotting , Pollos , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Decorina , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular , Lumican , Peso Molecular

RESUMEN

Proteoglycan synthesis was measured in chick sclera at the onset of form-deprivation myopia, as well as in the period immediately following removal of the occluder. Two day-old chicks were monocularly form vision deprived for periods from one to ten days and proteoglycan synthesis was determined after placing posterior scleral buttons in organ culture and measuring 35SO4 incorporation into glycosaminoglycans. Following 24 hrs of form-deprivation, proteoglycan synthesis was 33% higher in myopic eyes as compared with paired control eyes. The rate of proteoglycan synthesis further increased to levels 83% higher than controls after four days of form-deprivation and remained elevated throughout the ten day period of deprivation. Removal of the occluder after 10 days of form-deprivation resulted in a rapid drop in the rate of proteoglycan synthesis to control levels within 24 hrs. Proteoglycan synthesis was also measured in scleral chondrocytes isolated from control and myopic eyes after 10 days of form-deprivation. Proteoglycan synthesis by chondrocytes from myopic eyes did not return to control levels until 48 hrs after plating. Since the rate of proteoglycan synthesis returns to control levels more quickly during the recovery period ex vivo than when scleral chondrocytes from myopic eyes are placed in cell culture, we suggest that a mechanism is present within the eye which rapidly lowers the rate of proteoglycan synthesis in response to form vision.

Asunto(s)

Cartílago/metabolismo , Miopía/fisiopatología , Proteoglicanos/biosíntesis , Esclerótica/metabolismo , Animales , Cartílago/fisiopatología , Células Cultivadas , Pollos , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Glucosamina/metabolismo , Glicosaminoglicanos/biosíntesis , Luz , Técnicas de Cultivo de Órganos , Esclerótica/fisiopatología , Privación Sensorial , Visión Monocular

RESUMEN

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/genética , Córnea/fisiología , ADN/genética , Dermatán Sulfato/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , Clonación Molecular , ADN/aislamiento & purificación , Decorina , Proteínas de la Matriz Extracelular , Biblioteca de Genes , Glicosilación , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico