In this study, we first generated and characterized a polyclonal antibody against unique domain of matrlin-2 and then used this specific antibody to assess the expression pattern of matrilin-2 by immunohistochemistry. We found that marilin-2 is widely distributed in the connective tissues of many mouse tissues including heart, colon, penis, esophagus, lung, kidney, tracheal cartilage, developmental bone, and adult bone. The expression level of matrilin-2 was remarkably increased in the tissues of osteoarthritis developmental articular cartilage, compared to normal healthy tissues. Furthermore, we determined matrilin-2 expression in specific epithelial cells in stomach and ductal epithelial cells of salivary gland. In other tissues, the positive signals were mainly located around cardiac muscle cells and Purkinje fibers in the heart; corpus spongiosum in the penis; submucosa in the colon and esophagus; extracellular matrix of cartilage in the tracheal cartilage; and, glomerulus, the basement membrane of distal convoluted tubule and renal matrix in kidney. These observations indicated that the distribution pattern of matrilin-2 is heterogeneous in each tissue. Matrilin-2 may play an important role in the communication of matrix to matrix and matrix to cells and will be used as a potential biomarker in the early stage of osteoarthritis of articular cartilage.
Extracellular matrix (ECM) is composed of a large number of secretary multiple domain proteins, which form a filamentous network to connect cell surface and other ECM molecules. ECM proteins mediate cell-matrix and matrix-matrix communication and thereby determine the histoarchitecture specific to every organ and provide cells with crucial information on migration, adhesion, and differentiation [
Although the knowledge about matrilin-2 functions is accumulating, it still limits at the present time. Matrilin-2 is believed to be a novel family member of filament-forming oligomeric adapter proteins that are involved in the development and homeostasis of the extracellular matrix network [
There are seven putative Smad-binding sites within human matrilin-2 promoter and exon I [
Matrilin-2 contains a unique domain between the second vWFA domain and the C-terminal coiled-coil domain with no sequence homology of other family members and known proteins [
Newborn C57BL/6 and healthy adult C57BL/6 (6-week-old) mice were anesthetized and then sacrificed with an intraperitoneal injection of phenobarbital. Various mouse tissues including lung, brain, tongue, larynx, pharynx, salivary gland, esophagus, stomach, small intestine, large intestine, lymph node, liver, heart, pituitary, thyroid and parathyroid gland, ovary, oviduct, vagina, prostate, epididymis, spleen, kidney, skin, ureter, and testis were harvested and fixed in 10% PBS-buffered formalin. These fixed materials were routinely processed and embedded in paraffin. Serial sections at thickness of 5
Knee joins fromsixadult C57BL/6 mice and three nine-month-old male adult Duncan-Hartley guinea pigs with osteoarthritis developments were harvested and decalcified by 10% EDTA solution. These treated materials were processed and embedded in paraffin. Longitudinal serial sections of bone at thickness of 5
Tibias tissues from three nine-month-old male adult Duncan-Hartley guinea pigs with osteoarthritis development were fixed in 70% ethanol and then processed for methyl methacrylate embedding. 5
The human osteoarthritis tissues were collected from clinic knee arthroplasty. The samples were decalcified and embedded in paraffin, and 5
A peptide SRSTQKLFHSTKSSGNPLEE corresponding to C-terminal of the unique domain of mouse matrilin-2 was synthesized. The antiserum was raised in a New Zealand white rabbit by standard methods. IgG fraction was isolated from serum with a protein A-Sepharose column (Pharmacia Amersham Biotech, Piscataway, NJ) on a Pharmacia fast-performance liquid chromatography system. The purification procedure was described in detail elsewhere [
To determine the specification of this antibody raised in this study, we prepared three recombinant constructs including mini-mouse matrilin-2, mini-matrilin-3, and matrilin-1 as described previously [
Schematic drawings of the domain structure of recombinant matrilins variants (a) and analysis recombinant matrilin products (b). The recombinant proteins of matrilins were separated on a 4–15% gel, blotted to a membrane, and incubated with antiserum matrilin-2 antibody (upper pattern, both long and short forms of matrilin-2); FLAG tag ((b) middle pattern, long, short, and deleted matrilin-2); V5 tag (bottom pattern, all forms of matrilin-2, matrilin-1, and matrilin-3). The Western blot results showed our antibody can detect two different isoforms of matrilin-2 ((b) lanes 1 and 2 upper). If genetic engineering deletion unique domain mutant of mini matrilin-2, the recombinant product would not be detectable by our matrilin-2 antibody ((b) lane 3 upper); this product can be recognized by tags ((b) FLAG tag, lane 3 middle, and V5 tag, lane 3 bottom). This antibody is no cross reaction with other matrilins, such as matrilin-1 ((b) lane 4) and matrilin-3 ((b) lane 5).
Matrilins constructs
Western blot
For the cell culture media, 5
For the newborn and adult mice knee joint, tissue extracts were prepared as described [
Total RNAs were extracted from six newborn and six healthy adult C57BL/6 mice knee joints using RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. For quantitative real-time PCR, the experiment was performed using QuantiTect SYBR green PCR Kit (Qiagen, Valencia, CA) with DNA Engine Opicon 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA). Primers used in amplification of target genes mRNA are as follows: matrilin-2 (forward, 5′-TGCCTCTGAGCCCATTGACAAG-3′; reverse, 5′-TATGTTGCACTGTTGGCTGGT-3′); 18S RNA (forward, 5′-CGGCTACCACATCCAAGGAA-3′; reverse, 5′-GCTGGAATTACCGCGGCT-3′). The 18S RNA was amplified at the same time and used as an internal control. The matrilin-2 mRNA level was normalized to housekeeping gene 18S RNA levels. The relative value of matrilin-2 mRNA was measured and calculated by computer software (PE ABI, Foster City, CA). The data were presented as mean ± SEM for six samples and analyzed using two-way analysis of variance. The level of matrilin-2 in newborn and adult mice knee joints was designated as 1. Statistical significance was taken at
Immunohistochemistry was carried out using the Avidin-Biotin Complex (ABC) methods (Vector Labs). Representative sections were deparaffinized and rehydrated through conventional methods. Endogenous peroxidase was blocked by treating the sections with 3% hydrogen peroxide in methanol for 30 min. Digested by bovine testicular hyaluoidase [
In order to investigate the distribution pattern of matrilin-2, a primary antibody was raised in this study. We synthesized a peptide corresponding to the C-terminal of the unique region of mouse matrilin-2. The unique domain is matrilin-2 specific, which does not exist in other matrilins and any known proteins. To ensure the specificity of this antibody, we use matrilin-2L, matrilin-2S, matrilin-2D, matrilin-1, and matrilin-3 transfected into cos-1 cell line. The Western blot results showed the antibody only recognizes expressed proteins from two isoforms of matrilin-2 which contains a unique domain (Figure
Our previous experiments and other studies have shown that matrilin-2 mRNA is widely distributed in several tested tissues by RT-PCR and Northern blot [
Matrilin-2 expression in mouse dense connective tissues (DCT), loose connective tissues (LCT), and some specific epithelial cells: immunohistochemistry was performed by our unique domain-specific antibody. The localization of matrilin-2 protein was found in DCT, LCT, and some specific epithelial cells of a lot of organs. Immunostaining signal at least was detected in connective tissues of heart (a), colon (b), penis (c), esophagus (d), lung (e), kidney (f), and tracheal cartilage (g). We also found some specific epithelial cells strongly expressing matrilin-2 such as stomach (h) and duct epithelial cells of salivary gland (i). Bar, 200
Matrilins are first isolated from cartilage and are the inherent molecules of skeletal tissues [
Inherent expression of matrilin-2 in cartilage and bone. Newborn and adult mouse tibiae were investigated by immunohistochemistry. The positive signals localized at the cartilage of newborn mouse tibia including resting, proliferating, hypertrophic zone, and perichondrium, periosteum, new bone marrow, and ligaments (a and b). The strongest signal localized in hypertrophic chondrocytes. In the adult mice, matrilin-2 expression level is much decreased. The positive signal mainly located in articular cartilage and hypertrophic chondrocytes (c, d, and e). There are no positive signals detectable in other zones of cartilage and bone marrow. Bar, 350
By quantitative real-time RT-PCR, we determine the relative mRNA abundance from 6 newborn and 6 adult mice knee joints. In the adult mice, the relative mRNA abundance of matrilin-2 is
Comparisons the matrilin-2 mRNA and protein expression between newborn and adult mouse knee. Newborn and adult mouse knee were investigated by quantitative real-time RT-PCR (a) and Western blot (b). There is plenty of matrilin-2 mRNA in newborn mouse knee; the level of mRNA is significantly decreased (
Osteoarthritis (OA) is one of the most common and disabling diseases in the elderly, affecting nearly 80% of individuals older than 75 years [
Goldner staining showed damage of articular cartilage in an osteoarthritis (OA) developmental animal model of Duncan-Hartley guinea pigs. OA developmental animal model of Duncan-Hartley guinea pigs tibiae was investigated by Goldner staining. The Goldner staining showed there was damage in articular cartilage. (a) is low magnification; (b) is higher magnification. Bar, 1.5 mm in (a); 100
Matrilin-2 expression in Duncan-Hartley guinea pigs tibiae. OA developmental animal model of Duncan-Hartley guinea pigs tibiae had been investigated by immunohistochemistry. The positive signal mainly located in articular cartilage and hypertrophic chondrocytes (a–d). There are no positive signals detectable in other zones of cartilage and bone marrow. Bar, 1 mm in (a); 100
Matrilin-2 expression in total knee arthroplasty. In total knee arthroplasty samples, matrilin-2 is strongly located in extracellular matrix of the surface of joint (a) and chondrocytes of articular cartilage (b). Bar, 100
Matrilins are of multiple domain proteins [
The matrilin family contains four members, that is, matrilins 1, 2, 3, and 4. Matrilin-1 and matrilin-3 are cartilage-specific proteins, and their biological functions have widely been investigated. Matrilin-1 is involved in a variety of inherited chondrodysplasia [
Although Klatt et al. [
In the nonskeletal tissues, the expression of matrilin-2 mainly occurs around cardiac muscle cells and Purkinje fibers in the heart; around corpus spongiosum in the penis; submucosa in the colon and esophagus; extracellular matrix of cartilage in the tracheal cartilage; and, glomerulus, the basement membrane of distal convoluted tubule and renal matrix in kidney. The observations of this study demonstrate that matrilin-2 expressed in skeleton, dense and loose connective tissues, and some specialized epithelia in mice, which consists of a large filamentous network in the body. Furthermore, matrilin-2 acts as an adapter molecule connecting other proteins and proteoglycans in the extracellular matrix and plays an important role in the communication or balance between the extracellular matrix and epithelial cells.
Osteoarthritis (OA) is affecting nearly 80% of individuals older than 75 years and is one of the most common and disabling diseases in the senior population [
In summary, we have generated a unique domain antibody of matrilin-2 that can specifically recognize both long and short forms of matrilin-2 without cross reaction with matrilin-1 and matrilin-3. With this unique antibody, we examined the pattern of matrilin-2 expression in skeletal and nonskeletal tissues and found that matrilin-2 was widely expressed in many tissues and organs that may hint biological functional heterogeneity of matrilin-2. Moreover, we found that matrilin-2 may be a potential early biomarker of OA developmental articular cartilage.
Reverse transcription-polymerase chain reaction
Extracelluar matrix
von Willebrand factor A
Epidermal growth factor
Dense connective tissue
Loose connective tissue
Cartilage matrix protein
Osteoarthritis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Shukun Zhang, Honggang Liu, Jingshi Liu, and Junming Luo contributed to the study design and drafted the paper, Shukun Zhang, Yan Guo, and Jinwu Peng carried out experiments, Sara Javidiparsijani and Guirong Wang wrote the paper and provided important suggestions, and Junming Luo wrote the paper. All authors approved the final paper.
The authors thank Dr. Qian Chen (Rhode Island Hospital at Brown University, RI) and Dr. Yue Zhang (Hershey Medical Center of Pennsylvania State University, PA) for providing matrilin-1 and mini-matrilin-3 constructs.