Proteomics is a very useful method for studying biological development. In this study, we used label-free proteomics technology to identify the proteins of antler in the differential development stages and obtained a comprehensive proteomic data of sika deer antlers, containing 1,937 proteins with high degree of credibility. The data will provide valuable clues for understanding the molecular mechanisms of antler rapid growth and ossification.
First, we analyzed the antler core proteins. Among that, 132 proteins were absent in costal cartilage and the to 10 hub proteins were involved in gene expression regulation. CTNNB1 is a key downstream component of the classical Wnt signaling pathway, which plays a key role in promoting cartilage formation and bone formation during antler development [9, 16, 17]. Some studies have shown that CTNNB1 together with TCF proteins regulates osteoblast expression of osteoprotegerin, an important inhibitor of osteoclast differentiation . The loss of CTNNB1 in osteoblasts leads to osteopenia and stabilization of CTNNB1 leads to high bone mass. The antler-special proteins indicated that antler had a unique developmental mechanism different from costal cartilage.
Subsequently, we screened the antler period-specific proteins and significant differential expression protein. The results found that the expression pattern of these proteins has a distinct feature: in the growth phase, more protein expression is up-regulated. This unbalanced pattern has been revealed by previous quantitative proteomic analysis of cartilage formation . Undifferentiated cells express mixed genes making the biological functions of these upregulated proteins more complicated compared with chondrogenic cells. Golan-Mashiach also reported that stem cells express promiscuous genes and have many differentiation options . However, in the differentiated state, the expression of most genes decreases but expression of some specific genes increases. Therefore, the expression of cell protein may be related to its differentiation state.
In addition, collagen alpha-1(II) chain and collagen alpha-2(XI) chain, the main components and characteristics of mature chondrocytes, were up-regulated in the growth phase of antler. Previous study showed that type II collagen was slightly expressed in young antlers (6 weeks) but absent in old antlers . Hyaluronan and proteoglycan link protein 1 (HAPLN1) with the highest relative abundance of special proteins for antler growth stages is also called cartilage-associated protein 1 (CRTL1). Hyaluronic acid is an important extracellular matrix in the process of cartilage formation. HAPLN1 stabilizes the aggregation of proteoglycan and hyaluronic acid by binding the hyaluronic acid chain in aggrecan. HAPLN1 may also be used as a growth factor to up-regulate the synthesis of aggrecan and type II collagen in cartilage . Besides, two main enzymes were identified which are involved in the post-translational modification of chondrocyte ECM components, PAPSS1 and PAPSS2. In chondrocytes, the sulfation of proteoglycans is an important post-translational modification. In mammals, PAPSS plays an important role in the development of cartilage proteoglycan sulfation and adapts to the strong ECM synthesis during cartilage formation . Therefore, it can be considered that chondrogenic differentiation may be the main characteristic of the rapid growth of antler.
The upregulated proteins during antler ossification were significantly enriched in lysosome, acidic membrane-bound organelle that contain proteins required for osteoclast bone resorptive function . There is considerable evidence to suggest that Cathepsin B and cathepsin D are located in the lysosomes of osteoclasts [25–27] and the inhibitors have marked inhibitory effect on bone resorption capacity . Matrix metalloproteinases are a family of proteolytic enzymes that contribute to the degradation of the organic matrix of bones . MMP9 is highly expressed in bone cells especially osteoclasts and regulates ECM degradation and bone remodeling. The MMP9 null mice showed improved connectivity density of the trabeculae . The tartrate acid-resistant phosphatase (TRAP), which is commonly used as a specific marker of differentiated osteoclasts, is another lysosomal enzyme implicated in osteoclast resorptive function. It should be noted that the proteolytic enzymes generally require an acidic environment to be activated. Carbonic anhydrase II (CA II) is a zinc-containing metal enzyme that catalyzes the production of protons intracellularly from carbon dioxide . These protons produced are transported by vacuolar H+-ATPases (V-ATPases) through the ruffled border cell membrane into the resorption lacuna . ATP6V1A, a catalytic subunit of V-ATPases, is highly expressed in osteoclasts. Horng reported that knockdown of ATP6V1A can impairs acid secretion and ion balance in zebrafish . The up-regulation of lysosomal-related proteins suggests that the bone remodeling activity of deer antler in the ossification stage is higher than that in the growth stage.
Bone growth and the immune system are believed to have a close interaction. Many immune factors affect the differentiation and bone resorption of osteoclasts by regulating the RANK/RANKL/OPG system such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8. In this study, some immune-related molecules, such as Protein S100-A7 (S100A7), cathelicidin-7 (CATHL7), lactotransferrin (LTF), azurocidin (AZU1), neutrophil elastase (ELANE) and myeloperoxidase (MPO), were found to be up-regulated during ossification. These immune-related molecules play an important role in the regulation of osteoblast and osteoclast differentiation. S100A7 promotes osteoclast differentiation and activity by enhancing the effects of M-CSF and RANKL [34–36]. However, several other factors effectively promote the proliferation and differentiation of osteoblasts and bone formation while inhibiting bone resorption [37–44]. SHAO also found that immune-related factors are related to the process of intervertebral disc ossification when analyzing the differentially expressed genes in herniated discs with or without calcification . Therefore, we speculate that the local immune system may contribute to the ossification of antler tip.
In summary, this study used label-free proteomics to analyze the protein expression profiles of sika deer antlers in different developmental stages. This will provide valuable information for studying the molecular mechanism of antler rapid growth and ossification.