Expression of immunoregulatory tumor necrosis factor-like molecule TL1A in chicken chondrocyte differentiation

Abstract

The proinflammatory cytokine tumor necrosis factor (TNF) alpha is not encoded in the chicken genome. However, 1 member of the TNF family, TNF-like molecule 1A (TL1A), which is an important immunoregulatory protein, has recently been characterized in chickens. In this study, chicken TL1A (chTL1A) and 1 of its receptors, decoy receptor 3 (DcR3) were found to be expressed in developing bone of 14.5-day-old chicken embryos. Chicken chondrocytes were shown to express TL1A by polymerase chain reaction (PCR) amplification of cDNA and by immunohistochemical studies. Tissue expression was localized to the epiphyeal region of tubular bones, particularly cells of the epiphyseal plate, the outer chondrocytes of the cartilage-interfacing synovia, most of the synovial cells, and the stromal fibroblastic cells of the vascular channels of the femoral head. A tissue-specific developmental function of TL1A was supported by the presence of DcR3 in the embryonic connective tissue.

Introduction

Intensive research on the regulation of immune functions and recent genome projects identified nearly 2 dozen tumor necrosis factor (TNF) homologues in vertebrates, establishing the TNF superfamily of proteins (1). Corresponding receptors constitute the TNFR superfamily, which has an with even larger number of proteins (2,3). Ligands and receptors of these superfamilies have unique structural attributes, and they activate sophisticated signaling pathways for apoptosis, survival, proliferation, differentiation, and activation. Recent studies of TNF and TNFR gene families in the chicken are very limited. Chicken orthologues of TNFR members Edar, Troy, and Xedar, varying in homology with the corresponding mammalian proteins, were shown to be involved in skin and feather differentiation (4). The TNF family member B-cell-activating factor (BAFF) was implicated in chicken B-cell differentiation and survival (5). The TNF-related apoptosis-inducing ligand (TRAIL), TNFR-associated factor (TRAF), and TNFRII have been found to be expressed in lymphoid organs and to regulate immune response (6,7). Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TNF-like molecule 1A (TL1A), has been implicated in osteoclast formation: transgenic mice overexpressing DcR3 had significantly lower final bone differentiation compared with wild-type animals (6). One study found that TL1A was expressed during bone remodeling in the mouse (8); however, the function of this protein was not analyzed. We hypothesized that TL1A may contribute to the regulation of cartilage vascularization during differentiation. Since mature bone is poorly vascularized, TL1A may function as a limiting factor in angiogenesis during the final stage of embryonal ossification. Earlier stages of this process require intensive nutrient and oxygen supply; therefore, DcR3, a recptor for TLIA, may counteract with the antiangiogenic function of TL1A.

This study investigated the expression of TL1A and DcR3 in bone differentiation.

Materials and methods

Search by tBlastX for a putative TNF superfamily member in the chicken

A tBlastX analysis (with BLOSUM45) was performed on a free database of expressed sequence tags (ESTs) of chicken cDNA (http://www.chickest.udel.edu/) (7); rainbow trout and human (P01375) TNF proteins sequence were used for the query. Hits with greatest homology were selected as targets for polymerase chain reaction (PCR) cloning. The PCR primers were designed to amplify a core segment of the identified ChEST230f6 clone (EST 603232619F1) 474 base pairs (bp) long. In silico extension of the identified chicken EST clone was performed to achieve the full-length cDNA for the functional chTL1A.

Exon–intron boundaries were mapped by blasting and aligning the assembled cDNA sequence on chromosome contigs of the http://www.genome.wustl.edu databank. The clustered chromosomal position of chTL1A and other members of the TNF superfamily was also investigated according to techniques described previously (9).

Chicken DcR3 was identified in the same public database. Specific primers were designed to amplify a substantial portion of the published cDNA sequence (AY251406), and the resulting PCR fragment was cloned and sequenced.

Production and PCR amplification of cDNA

The chicken chTL1A and chDcR3 cDNA were amplified by reverse-transcription (RT) PCR from RNA isolated from chicken embryonic chondrocytes and cultured for 48 h, according to the method of Puskás et al (10), with the use of sense primer 5′-GCCGTGCTGCTCTGCCTGCT-3′ and antisense primer 5′-CACTGAGGGTCTTGGTGCTGGTCAG-3′. Cloning was performed with the use of sense primer 5′-GCAGCTCCCCACCCACGTAC-3 ′ and antisense primer 5′-AACCCAACTTCCACGACGCCG-3′. Amplicons were cloned into pGEM-TEasy TA cloning vector (Promega Corporation, Madison, Wisconsin, USA) and transformed into competent XL-1Blue bacteria (Stratagene, Cedar Creek, Texas, USA). Plasmid minipreps were sequenced with an ABI PRISM 7700 automatic sequencer (PE Biosystems, Foster City, California, USA).

Construction of a chTL1A expression vector

The extracellular part of the chTL1A gene was amplified from the cDNA mixture with 2 primers: 5′-CGGGATCCGCCGTGC TGCTCTGCCTGCT-3′, which contains a BamHI site (underlined), and 5′-CCGCTCGAGCCACAAAGCTGCTGCCAGAGACCTACAG-3′, which contains an XhoI site (underlined). For PCR, Pfu DNA polymerase (Fermentas, Vilnius, Lithuania) was used under the following conditions: denaturation for 3 min at 94°C; denaturation for 30 s at 93°C, annealing for 30 s at 55°C, extension for 1 min at 72°C for 35 cycles, and a final extension for 10 min at 72°C. The amplified product was checked on a 2% agarose gel and then isolated by means of an E.Z.N.A Gel Extraction kit (Omega Bio-Tek, Norcross, Georgia, USA). The isolated PCR fragment was digested with BamHI and XhoI and then ligated to corresponding sites of the His6-tag-encoding expression plasmid vector pFastBac HTb (Invitrogen SA, Merelbeke, Belgium). The Sf9 cell-line-producing chTL1A protein was established with the Bac-To-Bac system (Invitrogen) according to the manufacturer’s instructions.

Expression and purification of the recombinant protein

Insect cells (5 × 10⁷) containing the recombinant virus were grown in Insect Media (Sigma-Aldrich), according to the manufacturer’s instructions. Cells were collected by centrifugation and washed with phosphate-buffered saline. The pellet was lysed in 8 M urea, 150 mM NaCl, and 50 mM Tris-HCl (pH 8.0), kept on ice, and then centrifuged in a Beckmann JA-20 rotor at 3750 × g for 20 min. The supernatant was loaded onto a His6-affinity matrix (TALON; Clontech Laboratories, Palo Alto, California, USA) fast-performance liquid chromatogrpahy column of 1 mL bed volume, pre-equilibrated with the lysis buffer. After 5 bed volumes of washing with the loading buffer, a linear gradient of 0 to 150 mM of imidazole was applied to the column in the same buffer and staining with Coomassic Brilliant Blue was done. Fractions of 0.5 mL were collected and checked by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Serra Electrophoresis, Heidelberg, Germany).

Western blotting of recombinant chTL1A

Western blotting was carried out according to standard protocols on a nitrocellulose membrane. Briefly, 15-μL samples of crude extracts of insect cell lysates and fractions from the affinity chromatography were mixed with concentrated (2×) SDS Sample Buffer (Roche Applied Science, Indianapolis, Indiana, USA) and loaded onto a 15% SDS-PAGE gel; electrophoresis was run in a minitank at 150 V and 35 mA. The gel proteins were then electroblotted onto a nitrocellulose membrane in a plastic manifold at a constant 31-mA current at 4°C for 16 h in a buffer of 25 mM Tris, 192 mM glycin (pH 8.3), and 15% methanol. The membrane was washed in distilled water for 5 min on a rotary shaker and then saturated in a 5% solution of fatty milk powder dissolved in Tris-buffered saline for 1 h at 4°C. Primary and secondary antibodies [goat antibody P18 against TL1A (Santa Cruz Biotechnology, Santa, Cruz, California, USA)] and F-fragment specific peroxidase-conjugated rabbit IgG against goat antigen (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA), code: 305-036-008, respectively were applied to the filter according to the recommendations of the manufacturers. Peroxidase activity was detected with a SuperSignalWestPico chemiluminescence kit (Pierce Chemical, Rockford, Illinois, USA).

Immunohistochemical testing

Standard immunohistochemical detection of TL1A was performed on formalin-fixed, paraffin-embedded femoral bone of 14.5-d-old chicken embryos with the use of goat antibody P18 against VEGI (Santa Cruz Biotechnology, CA, USA) and immunoglobulin (Ig) against goat antigen followed by EnVision ± Labeled Polymer-HRP (horseradish peroxidase) conjugated antibody against rabbit antigen (Dako).

Native frozen sections were stained with rabbit antibody against mouse laminin (Dako) and visualized with donkey antibody against rabbit Ig-Cy2 or, for TL1A, donkey antibody against goat Ig-Cy3 (Jackson ImmunoResearch Laboratories).

Results

The tBlastX analysis of the chicken EST database with Rainbow trout and human TNF protein sequences for the query led to the identification of a clone containing a sequence encoding a polypeptide with high similarity to human VEGI/TL1A: ChEST230f6. With several overlapping chicken ESTs from different tissues assembled, the potential translational start and stop codons were located and the resulting cDNA was identical to the recently described full chicken TL1A (9). We deposited the cDNA sequence in GenBank (as AY954626).

Comparative genomic analysis showed that the chromosomal locus containing human TNF/LTαβ was completely lost in the chicken; however, traces of TNF-like cytotoxic bioactivity were detected in certain chicken tissues. A recently published paper has suggested that chTL1A might be the missing link between poultry and mammals (9). The gene for chTL1A was located on chromosome 17, in close proximity to CD30L, another member of the TNF superfamily (Figure 1A). These 2 genes were also located side by side on 1 chromosome in mammals (chromosome 9 in the human, chromosome 4 in the mouse), in accordance with a chicken–man–mouse linkage map (11). Moreover, the chTL1A gene spanned a segment 13 kbp long that had an exon–intron structure similar to that of mammalian TL1A (Figure 1B). These similarities between the poultry and mammalian genes indicate an evolutionary conservation of TL1A.

Figure 1.

A — Localization of the tumor nectorisis factor (TNF) superfamily gene cluster containing the gene for the TNF-like molecule A (TL1A) in chicken and mammals. B — Exons are represented as black boxes, introns as solid lines.

The chicken TL1A cDNA isolated from chondrocytic cells was expressed as baculoviral recombinant protein. It migrated in SDS-PAGE as a 26-kDa protein, similar to the mammalian protein, and was identified as TL1A by Western blotting (Figure 2). Nonimmune goat serum used as a negative control did not give a background signal (not shown).

Figure 2.

Results of 15% sodium dodecyl sulfate polyacrylamide gel electrophorsis of baculovirus-expressed recombinant chicken TL1A (see Materials and methods for details). A — after staining with Coomassie Brilliant Blue. B — after Western blotting with antibody against TL1A.

The immunohistochemical studies (Figure 3) showed that the chTL1A immunoreactivity was restricted to chondrocytes in bones of developing chicken embryos, especially around vascular structures. Specific immunostaining was found in the cells of the epiphyseal plate. The basal membrane of the vascular endothelium was visualized with laminin staining. The endothelium was clearly distinguished from proliferating chondrocytes by staining for TL1A and counterstaining with DAPI. The specific expression of the chTL1A gene product in proliferating chondrocytes suggests its role in bone development. Nonimmune goat serum used as a negative control again did not give a background signal (not shown).

Figure 3.

Expression of chicken TL1A on tissue sections of growth discs. A — Chicken knee articular section stained with hematoxylin and eosin. B to D — Epiphyseal cartilage stained with goat antibody against VEGI, immunoglobulin (Ig) against goat antigen, and horseradish peroxidase-conjugated antibody against rabbit antigen. E — Basal membrane of vascular endothelium stained with rabbit antibody against mouse laminen and visualized with donkey antibody against rabbit Ig conjugated with Cy2 (green). F — Cartilage stained with goat antibody against TL1A and visualized with donkey antibody against goat Ig-Cy3 (red). G. DAPI counter-staining of chondrocytes. H — Overlay of E, F, and G.

Chicken DcR3 was detected by expressional cloning in poultry chondrocytes (Figure 4). A 339-bp cDNA fragment was successfully cloned, and sequence analysis proved to be identical to the chDcR3 gene.

Figure 4.

Isolated polymerase chain reaction fragment with the expected size of 339 base pairs (bp) from the chicken chondrocyte cDNA pool cloned further in a vector and then sequenced. The sequence data proved that the fragment was identical to chicken decoy receptor 3 (chDcR3). MW — molecular weight.

Discussion

Cytokines have been shown to regulate cartilage differentiation and regeneration (12); however, data on the endogenous expression of these factors in cartilage and their function in differentiation and repair are limited. Kokubu et al (13) showed that the cytokines IL-17A and IL-17B, along with the IL-17 receptor and the IL-17 receptor-like protein are immunolocalized in bone during fracture repair. Types A, B, C, and D of vascular endothelial growth factor (VEGF) are crucial in vascularization during endochondral ossification, and have been found to be expressed in primary chondrocytes and ATDC5 chondrogenic cells (14). Other factors, including bone fibroblast growth factor (FGF), transforming growth factor β1 (TGF-β1), and VEGF, were found to be important in chondrocytes and eroded cartilage surface in Kashin–Beck disease (15). Most of these studies were carried out in mammals. However, the role of TGF-β in chondrocyte differentiation (16) and the upregulation of osteopontin by FGF signaling (17) were also shown in chickens. The TL1A was found to be expressed during bone remodeling in mice (18). The role of the TL1A receptor DcR3 in differentiation (8), immunomodulation (19), and angiogenesis (20,21) in mammals has been shown. A recent study of cloned TL1A proved the immunoregulating role of this factor in chickens (9), and another study identified the function of DcR3 in angiogenesis in chickens (21). Nevertheless, neither TL1A nor DcR3 expression during cartilage differentiation had yet been identified in chickens.

In this study, we cloned TL1A from chicken chondrocytes, proving it to be identical to the published chTL1A by sequence analysis and Western blotting. Immunohistochemical study verified the presence of TL1A in differentiating chondrocytes: it was present in cells of the epiphyseal plate, in the outer chondrocytes of the cartilage-interfacing synovia, in most of the synovial cells and in the stromal fibroblastic cells of the vascular channels of the femoral head. These results strongly suggest a role of this cytokine in bone differentiation and are supported by detection of the TL1A receptor DcR3 in embryonic connective tissue. These novel data may help studies in mammals as well; however, determining the role of TL1A and DcR3 in bone development and differentiation requires further studies.