Protein synthesis in secretory cells is episodically regulated to meet the rapidly changing demands for secreted proteins that participate in physiological and developmental processes. A prime example of episodic regulation of protein synthesis is the endocrine and exocrine pancreas, which responds within minutes of nutritional stimulation to secrete a panoply of stored peptide hormones and digestive enzymes. In the wake of secretion, synthesis of secretory proteins is activated to replenish the pools of stored proteins released from secretory granules (11
). As secretion subsides and the newly synthesized secretory proteins reach a critical concentration in the secretory granules, Golgi, and rough endoplasmic reticulum (RER), the synthesis of these proteins is repressed.
In the endocrine pancreas, the regulation of insulin secretion by the beta cells of the islets of Langerhans continues to be one of the most intensely investigated regulatory processes (30
). The ingestion of glucose stimulates insulin secretion within a few minutes and results in rapid insulin-induced glucose import into most cells of the body. Following induction of insulin secretion, both global protein synthesis and preproinsulin synthesis are derepressed from the relatively low levels observed in the fasted state. The regulation of protein synthesis and preproinsulin synthesis is poorly understood. Translational control exerted through the regulation of mRNA cap binding proteins and the regulation of the eukaryotic initiation factor 2 (eIF-2)-GTP-Met-tRNAi ternary complex have been implicated in the regulation of protein synthesis in the insulin-secreting beta cells, but definitive biochemical and genetic analysis is lacking. The exocrine pancreas is stimulated to secrete a complex of diverse digestive enzymes by a variety of stimuli, including acetylcholine, gastrin, and cholecystokinin (30
). Despite the fact that cotranslational import of proteins into the ER was first discovered and extensively characterized in the pancreatic acinar cells (10
), the fundamental mechanisms underlying the highly dynamic regulation of protein synthesis in the exocrine pancreas have not been elucidated.
Similar to pancreatic cells in their extraordinary secretory capacity are chondrocytes and osteoblasts, the major secretory cells of the skeletal system, which are responsible for the massive secretion of the cartilage and bone matrix proteins that occurs during prenatal and postnatal development. The synthesis and secretion of collagen and other extracellular proteins that comprise cartilage and bone matrix are dynamically regulated as a consequence of the complexity of the growth of the skeletal system, as well as changes in serum levels of growth factors, calcium, and nutrients.
Clues to the regulation of protein synthesis in secretory cells have recently come from the investigation of the unfolded protein response (UPR) (3
), an adaptive mechanism to thwart the deleterious effects of accumulating toxic concentrations of unfolded proteins in the ER. The UPR in higher eukaryotes involves transcriptional activation of the folding and chaperone proteins along with repression of global protein synthesis. Recently, an eIF-2α kinase, denoted PERK/PEK, has been discovered and is responsible for UPR-induced repression of protein synthesis (14
PERK is hyperactivated by pharmacological agents that disturb the Ca2+
balance, protein folding, or glycosylation in the ER (13
). Phosphorylation of eIF-2α by PERK under these extreme conditions results in global repression of protein synthesis. Among the eIF-2α kinase family, PERK is uniquely present in the ER as a type 1 transmembrane protein, and its activity is regulated by the BiP/GRP78 ER resident protein chaperone (3
). Through the interaction of BiP/GRP78 with the ER lumenal domains of PERK and IRE1, it has been proposed that BiP/GRP78 negatively regulates PERK and IRE1 by blocking their ability to form oligomers or homodimers. When the folding capacity of the ER is exceeded, either by increasing the concentration of unfolded proteins or by decreasing the folding or chaperone proteins, BiP/GRP78 disassociates from PERK, allowing it to form activated homomeric complexes. Hyperactivated PERK results in the repression of global protein synthesis and may further result in apoptosis. Based upon these findings, PERK has been categorized as a stress-related protein. However, we have discovered that PERK is abundantly expressed in secretory and endocrine organs, including the exocrine and endocrine pancreas, as well as osteoblasts, which secrete type I collagen, the most abundant protein in the body. Although PERK is thought to be expressed at low levels in virtually all cells, the high level of expression in secretory cells suggested to us that PERK may have an important regulatory role in secretory cells under normal physiological conditions.
In addition to PERK, mammalian species contain three other eIF-2α kinases: GCN2, HRI, and PKR (2
). Each of the eIF-2α kinases is activated by different factors, but all specifically phosphorylate Ser51 of eIF-2α. Phosphorylated Ser51 eIF-2α, complexed with eIF-2β, eIF-2γ, and GDP, blocks the GDP-GTP exchange reaction by sequestering the eIF-2B guanylate exchange factor in an inactive state. In the absence of eIF-2B activity, the eIF-2-GTP-Met-tRNAi ternary complex cannot be replenished for subsequent rounds of translation initiation (6
). In the extreme, hyperphosphorylation of eIF-2α results in complete repression of global protein synthesis followed by cell death. However, more moderate levels of phosphorylated eIF-2α (eIF-2α[P]) can actually stimulate the translation initiation of specific mRNAs encoding regulatory proteins, as has been elegantly demonstrated for the yeast GCN4 transcriptional activator of amino acid biosynthesis (16
Mammalian cells typically contain a low level of eIF-2α[P]; however, we show herein that eIF-2α is highly phosphorylated in the pancreas by PERK during the fasted state. Upon glucose stimulation, eIF-2α is dephosphorylated over the same time course as the induction of insulin synthesis. These data further suggest that PERK may play an important role in regulating protein synthesis in secretory cells. To determine the role of PERK in regulating protein synthesis in secretory organs and tissues, we generated a knockout mutation of the mouse Perk gene. We show that a fraction of homozygous Perk mutants are viable but display a number of severe defects in pancreatic functions, are growth retarded, and exhibit multiple skeletal dysplasias. These defects are associated with major shifts in the synthesis of secretory proteins, misregulation of genes that function in protein secretion, gross abnormalities in the RER, and apoptotic cell death.