Search

Aim of our study is to provide an insight into the genetic expression landscape of GREB1L, ITGA10 and CRELD2 which are important in human genitourinary tract development which might help elucidate the critical stages for the onset of kidney anomalies.Morphological parameters were analyzed using immunohistochemistry on human foetal (13-38 w) and postnatal (1.5 and 7.5y) human kidney samples.GREB1L marker had a strong intensity and the highest rate in proximal tubules (PTC) of 1.5 years' kidney (90.25%). In the distal tubules (DCT) there were statistically significant differences in 13 w, 15 w, 16 w, 21 w, 38 w and 7.5y regarding 1.5y (Kruskal-Wallis test, p 0.001). There was significantly more GREB1L in the glomeruli at 21 w and 38 w in regard to all other stages (Kruskal-Wallis test, p 0.01). ITGA10 staining intensity was strongest in PCT with the highest rate in 13 w (92.75%), while the lowest rate was found in glomeruli and DCT (Kruskal-Wallis test, p 0.001). CRELD2 had the strongest staining intensity in PCT with the highest rate in 13 w and 1.5y (92.25%) and lowest in the glomeruli of 7.5 years (24.3 %). In DCT there were statistically significant differences in CRELD2 positive cells in 13 w, 15 w, 16 w, 21 w, 38 w and 7.5y regarding 1.5y (Kruskal-Wallis test, p 0.01). ITGA10 and CRELD2 co-localised in the postnatal period in DCT.High kidney expressions of GREB1L, ITGA10 and CRELD2 even in the postnatal period implicate their importance not only for the onset of CAKUT in the case of their mutation but also for maintenance of kidney homeostasis.

The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.

Cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER)-resident chaperone highly activated under ER stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage-specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whereas deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating noncanonical WNT4 signaling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor low-density lipoprotein receptor-related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF-β1 signaling. Therefore, our data provide a novel link between an ER-resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT-responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique headto-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study, we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5’-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reduced ALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in this study. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0193-y) contains supplementary material, which is available to authorized users.

In this study, we found that Cysteine-rich with EGF-like domains 2 (CRELD2), a novel endoplasmic reticulum stress-inducible protein, is not only localized in the ER-Golgi apparatus but also spontaneously secreted. Deletion of four C-terminal amino acids from mouse CRELD2 or addition of tag-peptides to its C-terminus dramatically enhanced CRELD2 secretion. Intra- and extra-cellular CRELD2 is differentially glycosylated and its spontaneous secretion was significantly prevented by overexpression of a dominant negative mutant Sar1 and treatment with brefeldin A. Overexpression of wild-type GRP78 remarkably enhanced the secretion of wild-type but not mutant CRELD2. Our results demonstrate both that CRELD2 is a novel secretory glycoprotein regulated by Sar1 and GRP78 and that the C-terminal of CRELD2 plays a crucial role in its secretion.