The parathyroid gland-specific proteome

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Figure 1. The distribution of all genes across the five categories based on transcript abundance in parathyroid gland as well as in all other tissues.

359 genes show some level of elevated expression in the parathyroid gland compared to other tissues. The three categories of genes with elevated expression in parathyroid gland compared to other organs are shown in Table 1. The function and cellular localization of known genes with tissue enriched expression in parathyroid gland (n=60), are well in-line with the function of the parathyroid gland. In Table 2, the 12 genes with the highest level of expression among 60 enriched genes are defined.

Table 2. The 12 genes with the highest level of enriched expression in parathyroid gland. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. "mRNA (tissue)" shows the transcript level as TPM values, TS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Some of the proteins predicted to be membrane-spanning are intracellular, e.g. in the Golgi or mitochondrial membranes, and some of the proteins predicted to be secreted can potentially be retained in a compartment belonging to the secretory pathway, such as the ER, or remain attached to the outer surface of the cell membrane by a GPI anchor.

The parathyroid gland transcriptome

An analysis of the expression levels of each gene made it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 78% of the mRNA molecules derived from parathyroid gland correspond to housekeeping genes and only 14% of the mRNA pool corresponds to genes categorized to be either parathyroid gland enriched, group enriched or, parathyroid gland enhanced. Thus, most of the transcriptional activity in the parathyroid gland relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.

Gene Ontology-based analysis of all the 359 genes elevated in parathyroid gland identifies receptor activity, molecular transducer activity and calcium ion binding as main functions. A majority of the 359 genes encode membrane-bound proteins.

Protein expression of genes elevated in parathyroid gland

In-depth analysis of the elevated genes in parathyroid gland using antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in different functional compartments including proteins involved in extracellular calcium homeostasis and proteins that bind intracellular calcium.

Proteins involved in extracellular calcium homeostasis in parathyroid gland

Parathyroid gland regulates calcium homeostasis by producing and releasing parathyroid hormone (PTH). Extracellular calcium level is continuously monitored by CASR, a receptor located at the cell membrane of chief cells. In response to decreased calcium level, PTH synthesis is triggered in chief cells whereupon it is released into the blood. It restores calcium levels in blood by binding parathyroid hormone receptor in bone and kidney which results in increased bone resorption and calcium reabsorption. Calcium reabsorption in kidney is also regulated by CASR, located at cell membrane of epithelial cells in the thick ascending limb in the loop of Henle.

Proteins that bind intracellular calcium in parathyroid gland

Several genes with an elevated expression in parathyroid gland encode proteins that bind intracellular calcium ions. CHGA is abundant in secretory granules and has high affinity for calcium ions but does not bind them strongly. Thus, CHGA facilitates storage of calcium ions in secretory granules and a buffering mechanism between these vesicles and the cytosol. PVALB binds calcium ions in the cytosol and is believed to be involved in the buffering of intracellular calcium. STXBP5 is a less characterized intracellular protein that may regulate calcium-dependent exocytosis by inhibiting membrane fusion between intracellular vesicles and cell membrane.

Genes shared between the parathyroid gland and other tissues

There are 63 group enriched genes expressed in the parathyroid gland. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including parathyroid gland, compared to all other tissues.

In order to illustrate the relation of parathyroid gland to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types.

Figure 2. An interactive network plot of the parathyroid gland enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of parathyroid gland enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Parathyroid gland shares group enriched genes mainly with cerebral cortex. A Gene Ontology based analysis of these shared genes shows that they are involved in various functions and processes including transporter activity, regulation of synapse organization and cell adhesion. One example of a protein expressed in both parathyroid gland and cerebral cortex is PEX5L, an intracellular protein that may regulate hyperpolarization-activated cyclic nucleotide-gated channels.

Parathyroid gland function

Parathyroid gland regulates calcium homeostasis by producing and releasing parathyroid hormone (PTH). PTH restores blood calcium levels by 1) activating osteoclasts to release calcium from bone into blood, 2) increasing calcium reabsorption in the kidney and 3) triggering activation of vitamin D in the kidney. The active form of vitamin D, calcitriol, stimulates uptake of calcium from food through the small intestines and further release of calcium from bone.

PTH concentration in the blood is dependent on the release of preformed PTH stored in secretory granules and by the synthesis of new PTH. The activity of calcium sensing receptor (CASR) regulates secretion of preformed PTH. This receptor is located at the cell membrane of chief cells and detects changes in extracellular calcium level by binding calcium ions. High extracellular calcium concentration activates CASR and consequently triggers an intracellular signal pathway that inhibits secretion of preformed PTH. Low calcium level has an opposite effect: PTH secretion is increased in the absence of CASR activation. Thus, the level of PTH in blood changes within minutes and the delicate balance in calcium homeostasis is maintained. Calcitriol alters the transcription of the PTH gene, thus affecting the replenishment of PTH stored in secretory granules. It also may have an indirect effect on PTH release by increasing the expression of CASR.

Parathyroid gland histology

Parathyroid glands are highly vascularized endocrine organs located behind the thyroid gland. There are typically four parathyroid glands, each about 5 mm in size and weighing 130 mg, however, the exact number and size of the glands may vary depending on individual. Chief cells are the predominant cell type characterised by round nucleus surrounded by scarce cytoplasm. They produce and secrete PTH in response to low extracellular calcium level detected by receptors in the cell membrane. Larger oxyphil cells with an eosinophilic cytoplasm and a slightly smaller nucleus form clusters scattered between chief cells. The function of oxyphil cells is still unknown and whether they are derived from or they are a deactivated form of chief cells is debated. A third cell type, transitional oxyphil cells are similar to size to chief cells but have a more eosinophilic staining, which may be an evidence of a transition from chief cells to oxyphil cells.

The histology of human parathyroid gland including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.

Background

Here, the protein-coding genes expressed in the parathyroid gland are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the parathyroid gland.

Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 1 fresh frozen tissues representing normal parathyroid gland was compared to 171 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in parathyroid gland. A tissue-specific score, defined as the ratio between mRNA levels in parathyroid gland compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression. These categories include: genes with elevated expression in parathyroid gland, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in parathyroid gland, and genes not expressed in any tissue. Genes with elevated expression in parathyroid gland were further sub-categorized as i) genes with enriched expression in parathyroid gland, ii) genes with group enriched expression including parathyroid gland and iii) genes with enhanced expression in parathyroid gland.

Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.

Relevant links and publications

Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed: 25613900 DOI: 10.1126/science.1260419

Yu NY et al, 2015. Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res.
PubMed: 26117540 DOI: 10.1093/nar/gkv608

Fagerberg L et al, 2014. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics.
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600

Kumar R et al, 2011. The regulation of parathyroid hormone secretion and synthesis. J Am Soc Nephrol.
PubMed: 21164021 DOI: 10.1681/ASN.2010020186

Ritter CS et al, 2012. Differential gene expression by oxyphil and chief cells of human parathyroid glands. J Clin Endocrinol Metab.
PubMed: 22585091 DOI: 10.1210/jc.2011-3366

D'amico MA et al, 2014. Biological function and clinical relevance of chromogranin A and derived peptides. Endocr Connect.
PubMed: 24671122 DOI: 10.1530/EC-14-0027

Histology dictionary - the parathyroid gland