Expression of the Discoidin Domain Receptor Family Depended on Glucose and Their High Expression in Arterial Tissues in the Rat Model of Type 2 Diabetes

Abstract

The active form of discoidin domain receptors (DDRs) is expressed in cell surface and regulated post-translationally by glucose. The DDR2 and DDR1 transfected in HEK293 cells were expressed mainly in their active forms with sizes of 130 and 120 kDa, respectively. DDRs were observed predominantly as 100 kDa proteins in glucose-depleted culture conditions. However, transfection of endothelial growth factor receptor (EGFR) in HEK293 cells resulted in the expression of only one form regardless of glucose concentration. Vascular smooth muscle cells, HT1080s, and MDA-MB-231 cancer cells expressed DDRs in their active forms in high glucose concentrations, which did not occur with EGFR. In diabetic rats, DDRs were expressed at high levels in arterial tissue but EGFR was not highly expressed. Taken together, these results suggest that DDRs expression depends on glucose concentration it may cooperate in the development of atherosclerosis and kidney fibroblasts, promoting nephropathy in diabetic rats.

INTRODUCTION

The discoidin domain receptors (DDRs), including DDR1 and DDR2, belong to the receptor tyrosine kinase (RTK) family and recognize native forms of various collagens as their activating ligands.^1–3) The extracellular regions of DDR proteins contain two structural units, one called the discoidin (DS) domain and another called the DS-like domain because of its structural similarity to the DS domain.⁴⁾ The DS domain, a stretch of approximately 160 amino acids, is responsible for collagen binding, and the DS-like domain plays a role in activating receptors upon ligand binding.^5,6) Previous work showed that the DS-like domain contains a calcium-binding site and two N-glycosylation sites that are strictly conserved in all members of the vertebrate DDR family.⁷⁾ Moreover, site-directed mutagenesis that abolished the conserved N-glycosylation site prevented DDR2 from completing its necessary posttranslational modifications, leading to DDR2 blockage within the endoplasmic reticulum (ER).⁸⁾ It has also been found that most missense mutations of DDR2 in human hereditary diseases caused defects in posttranslational modification and prevented trafficking of the protein to the cell membrane.^9,10)

DDRs are thought to play roles in the regulation of cell proliferation, adhesion, and migration of fibroblasts.^11,12) They also promote the remodeling of the extracellular matrix by inducing the expression of matrix metalloproteinases (MMPs) and the epithelial–mesenchymal transition.^13–16) DDR2 appears to be essential for proper bone growth. A knockout of the DDR2 gene is associated with dwarfism in mice, and a hereditary mutation in the coding region of DDR2 results in a shortened-limb phenotype in humans.^17–19) Elevated expression levels of DDR1 and DDR2 have been observed in vascular smooth muscle cells at atherosclerotic sites, and knockout of these genes is also associated with a phenotype that correlates with the development of renal fibrosis.^20–22) The expression of DDRs and point mutations in these proteins have been associated with various human cancers, such as lung, epithelial, ovarian, breast, and gastric cancers.^23–27)

Both DDR1 and DDR2 can be detected in the atherosclerotic plaques of nonhuman primates.²⁰⁾ Previous studies using Ddr1⁻/⁻ mice determined that DDR1 played a critical role in mediating neointimal hyperplasia after arterial injury, and Ddr1⁻/⁻ smooth muscle cells (SMCs) exhibited reduced MMP expression and proliferation and migration activities; overexpression of DDR1 rescued these deficits.^28,29) Diabetes mellitus, or simply diabetes, is a group of metabolic diseases in which individuals have high glucose concentrations in their blood and can develop atherosclerosis with high collagen expression.³⁰⁾ Both of these symptoms are related to the functions of DDRs. Using streptozotocin-treated female rats, a widely used model of type I diabetes, it has been shown that changes in the levels of arginase I, endothelial nitric oxide synthase, and cyclic guanosine monophosphate (cGMP)-dependent protein kinase play roles in modulating smooth muscle relaxation.^31–33) A recent study found that DDR2 functioned not only as a mediator of cardiovascular remodeling but also as a molecular target of metformin. Therefore, it has been suggested that DDR2 plays protective roles in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.³⁴⁾ In this study, we investigated the expression profiles of DDRs in transfected, primary, and cancer cells and evaluated glucose concentrations and arterial wall structures using a diabetic rat model.

MATERIALS AND METHODS

Ethics Statement

All animal studies were performed in strict accordance with the guidelines and principles established by the Korean Animal Protection Law (http://animalrightskorea.org). The use of animals was approved by the Institutional Ethics Committee for Animal Care of the Korean Institution of Science and Technology (Approval Number: KIST-5088-2022-05-080). All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) as adopted and promulgated by the National Institutes of Health.

Reagents, Antibodies, and Western Blotting

A polyclonal antibody against human DDR2 (AF2538) was purchased from R&D systems. Antibodies against DDR1 (C-20, rabbit polyclonal) and endothelial growth factor receptor (EGFR, sc-03, rabbit polyclonal) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). A glucose assay kit and SMCs (α-actin, A5228) were purchased from Sigma (St. Louis, MO, U.S.A.). Rompun was purchased from Bayer HealthCare Pharmaceuticals (Montville, NJ, U.S.A.). Zoletil 50 was obtained from Virbac (Carros, France). Western blotting was performed as described previously.⁸⁾ In brief, equal amounts of total cell lysates were subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and blotted to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.). The membranes were then blocked with 5% skim milk in Tris-buffered saline (TBS) buffer before being incubated with primary antibodies dissolved in 5% skim milk solution in TBS overnight at 4 °C. The resulting membranes were washed 5 times with TBS and applied with horseradish peroxidase (HRP)-conjugated secondary antibody. A chemiluminescence detection kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, U.K.) was used for signal detection.

Cell Lines and Cultures

HEK293 cells stably expressing either DDR1b (HEK293-DDR1), hemagglutinin (HA)-tagged DDR2 (HEK293-DDR2), or EGFR (HEK293-EGFR) were used.³⁵⁾ Primary rat vascular SMCs were prepared from 6-week-old rats as previously described.³⁶⁾ In brief, adherent fat and connective tissue were removed by blunt dissection. Vessels were then opened longitudinally and pre-incubated in Hank’s Balanced Salt Solution (HBSS) containing 1 mg/mL collagenase, 0.5 mg/mL elastase, 10% fetal bovine serum, and 0.25 mg/mL trypsin inhibitor (Type I-S: from soybean). The filtrate was collected and suspended in 10% fetal bovine serum (FBS) Dulbecco’s Modified Eagle Medium (DMEM). Cells were centrifuged (1500 rpm, 5 min), and the cell pellets were resuspended in 4 mL of 10% FBS DMEM. The cells were then cultured at 37 °C with 95% air and 5% CO₂. The human cancer cell lines MDA-MB-231 and HT1080 were obtained from American Type Culture Collection (ATCC) and maintained in Roswell Park Memorial Institute medium (RPMI) containing 20 mM glucose, 10% FBS, and 150 µg/mL penicillin–streptomycin (Gibco-BRL) in 5% CO₂ at 37 °C in a humidified incubator. When 5 mmol/L glucose- or glucose-free DMEM was used, 20 mM sodium pyruvate was added to the medium as a compensatory energy source.

Animal Experiments

Sprague–Dawley rats were obtained from Charles River (Constant, QC, Canada) and used to investigate the effects of diabetes on arterial walls. Animals were fasted for 18–24 h and then intraperitoneally administered nicotinamide (230 mg/kg) and streptozotocin (STZ; 65 mg/kg) in 0.02 M citrate saline buffer. The age-matched control group received citrate buffer only.³⁷⁾ Blood glucose levels were monitored every 2 weeks using a glucose assay kit (Sigma). Rats with blood glucose levels ≥15 mmol/L for 2 consecutive weeks were considered diabetic. Four weeks after the initial administration of streptozotocin or buffer, immunohistological evaluations of arterial tissue were conducted.³⁸⁾

Preparation of Serum from Whole Blood

Blood was incubated at 37 °C for 30–60 min to allow for clotting. After incubation, the rubber stopper was pulled off the top of the tube, and a long Pasteur pipette was used to “ring” the clot, i.e., separate it from the sides of the glass tube. The tubes were then placed at 4 °C overnight so that the clots could contract. Then, the serum was decanted away from the clot into a new centrifuge tube. The serum was then spun at 3000 rpm for 10 min to remove the remaining clots/red blood cells and other insoluble material, and the clarified serum was then stored at −80 °C.

Immunohistochemistry

For the immunohistological evaluation of arterial tissue, 5-µm-thick slices of tissue sections were deparaffinized with xylene and rehydrated, treated with 3% H₂O₂ for 5 min, and blocked with 1% normal serum. The slides were then incubated with antibodies against DDR2, DDR1, EGFR, and smooth muscle actin (SMA) followed by incubation with secondary antibodies (GenDEPOT, Baker, TX, U.S.A.). The staining signals on the tissues were developed according to the manufacturer’s instructions. All sections were photographed using a microscope. The amounts of stained levels were quantified by using ImageJ software.³⁹⁾

Statistical Analysis

Data from triplicate experiments were reported as means ± standard deviation, and n values represented the number of rats used. Statistical analysis for comparisons was performed by using one-way ANOVA. p-Value <0.05 was considered statistically significant. For comparison of body weight and glucose level between control and diabetic rats was analyzed using Student’s t-test in Microsoft Excel.

RESULTS

DDR Family Proteins Generated Multiple Isoforms Depending on Glucose Concentration

The 100-kDa DDR2 isoform was the predominant stable species after incubation of HEK293-DDR2 cells in glucose-free DMEM for 24 h (Fig. 1A, first lane). After 4 h, glucose-free DMEM was replaced with DMEM containing 20 mmol/L glucose, and two stable intermediate isoforms with molecular weights of 110 and 120 kDa were detected. The active isoform of DDR2 with a molecular weight of 130 kDa was expressed at the cell surface and became the dominant species 8 h after the glucose was added (Fig. 1A). The stepwise appearance of DDR2 isoforms with distinct molecular masses depending on glucose addition indicated the progression of the N-glycosylation process. Similar results were observed for DDR1 when at least three distinct isoform bands of approximately 100, 110, and 120 kDa in molecular weight were detected when HEK293-DDR1 cells in glucose-free DMEM were re-fed with 20 mmol/L glucose in DMEM (Fig. 1B). However, only one band of approximately 170 kDa was detected in HEK293-EGFR cells regardless of the glucose concentration in DMEM (Fig. 1C).

Fig. 1. Multiple Isoforms of DDRs Corresponding to Changes in Glucose Concentration Were Generated through Alterations in the N-Glycosylation Process

(A) HEK293-DDR2, (B) HEK293-DDR1, and (C) HEK293-EGFR cells were cultured in DMEM containing 20 mmol/L glucose after pre-incubation in glucose-free DMEM for 24 h. The total cell lysates were subjected to immunoblotting. (D) HEK293-DDR2, (E) HEK293-DDR1, and (F) HEK293-EGFR cells were cultured in DMEM containing an initial glucose concentration of 5.5 mmol/L and were harvested at different incubation durations for immunoblotting with DDR2, DDR1, and EGFR antibodies, respectively. (G–I) The glucose concentrations in the culture media of the three cell lines, HEK293-DDR2, HEK293-DDR1, and HEK293-EGFR, were assayed at different incubation times, respectively. The experiments were conducted in triplicate. Each column with a vertical bar represents the mean ± standard deviation from three experiments.

The same numbers of HEK293-DDR2, HEK293-DDR1, and HEK293-EGFR cells were cultured in DMEM containing 20 mmol/L glucose for 12 h, which was replaced with 5.5 mmol/L glucose in DMEM. The molecular mass changes of these RTKs were then determined by Western blotting. In HEK293-DDR2 cells, the active isoform of DDR2 (130 kDa) began to decrease during the 24 h period following the DMEM change, and the predominance of the 100 kDa isoform began to increase gradually after 24 h. At this point, the active isoform (130 kDa) had disappeared almost completely, and the 100 kDa protein was the major isoform detected (Fig. 1D). HEK293-DDR1 cells also showed a similar pattern of change in the predominant molecular masses of the isoforms of DDR1 in this experiment (Fig. 1E). In contrast, HEK293-EGFR cells exhibited almost no changes in the molecular mass isoform of EGFR (170 kDa) even after 48 h of incubation (Fig. 1F). The changing molecular weights of the DDR family were associated with a time-dependent decrease in the glucose concentration in the culture medium, which decreased below 0.1 mmol/L after 24 h (Figs. 1G, H, Supplementary Table S1). The change in the glucose concentration occurred almost identically in the HEK293-EGFR culture in DMEM (Fig. 1I, Supplementary Table S1).

In the next experiments, the molecular mass changes of the three proteins (DDR2, DDR1, EGFR) were compared in primary rat vascular SMCs cultured under glucose-depleted conditions. When vascular SMCs were maintained in a high-glucose medium (20 mmol/L), the 120-kDa isoforms were most abundant for both DDR2 and DDR1, but the 100 kDa isoforms became dominant after incubation of the cells in glucose-free medium for 24 h. In contrast, there was no significant change in the molecular weight of EGFR in the same experiment (Fig. 2A). We also evaluated the changes in the molecular masses of these proteins in two fibrosarcoma (HT1080) and breast (MDA-MB-231) cancer cell lines. After culturing equal numbers of these cancer cell lines in 5.5 mmol/L glucose in DMEM, glucose was almost depleted after 24 h in the culture medium. Among these RTKs, only DDR2 and DDR1 showed reductions in their molecular weights; in contrast, EGFR remained almost unaltered for up to 24 h (Figs. 2B, C). Taken together, these results suggest that the expression levels of the high-molecular-weight isoforms on the cell surface were selectively sensitive to glucose.

Fig. 2. DDR Family Proteins Underwent Highly Sensitive Molecular Weight Changes upon Glucose Depletion in Primary and Cancer Cell Lines

(A) Primary rat vascular smooth muscle cells incubated in glucose-free DMEM were harvested at different times for immunoblotting against DDR2, DDR1, and EGFR antibodies. (B) HT1080 cells cultured in RPMI and (C) MDA-MB-231 cells cultured in DMEM containing glucose at an initial concentration of 5.5 mmol/L were harvested at different incubation times and immunoblotted with DDR2, DDR1, and EGFR antibodies. The experiments were conducted in triplicate.

Blood Glucose Concentrations and Body Weights of Control and Diabetic Rats

Eight male Sprague–Dawley rats were used in this study. The fasting blood glucose levels before streptozotocin injection were 7.63 ± 0.18 and 7.69 ± 0.34 mmol/L in the control and diabetic groups, respectively. In the diabetic group, the glucose levels reached 28.46 ± 1.93 and 28.67 ± 2.53 mmol/L after 14 and 28 d, respectively, and the glucose levels in the control group were maintained at approximately 7.61 ± 0.19 mmol/L over the 28 d of the experiment (Fig. 3A, Table 1). After 14 d of streptozotocin injection, the blood glucose concentration was increased by approximately 370% in the diabetic group compared to the control group. The blood glucose levels in the diabetic rats remained elevated throughout the 28 d of the study, after which the experiment was terminated. Regarding changes in rat body weight, in the control group, after 28 d, the body weights increased 157% (from 222 ± 8.5 to 349 ± 8.0 g), and in the diabetic group, the body weights increased 139% (from 223 ± 7.5 to 311 ± 18.0 g) (Fig. 3B, Table 1).

Fig. 3. Blood Glucose Concentrations and Body Weights of Control and Diabetic Rats

Male Sprague–Dawley rats (6–8 weeks of age) were fasted for 18–24 h and administered 0.02 M citrate saline buffer (Control) or nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg) in citrate buffer (Diabetics) by intraperitoneal injection. (A) The blood glucose concentrations and (B) body weights of the control and diabetic groups were evaluated after 14 and 28 d. Each column with a vertical bar represents the mean ± standard deviation from 4 animals. * p = 0.0003, vs. control rat. ** p = 0.0004, vs. control rat. *** p = 0.0116, vs. control rat (Student’s t-test).

Table 1. Blood Glucose Concentrations and Body Weights of Control and Diabetic Rats via Time

	Blood glucose (mmol/L)			Body weight (g)
	0 d	14 d	28 d	0 d	14 d	28 d
CR-1	7.83	7.89	7.67	221	267	340
CR-2	7.67	7.56	7.39	225	276	359
CR-3	7.61	7.72	7.83	230	281	351
CR-4	7.39	7.33	7.56	210	289	346
CR Ave	7.63 ± 0.18	7.63 ± 0.24	7.61 ± 0.19	222 ± 8.5	278 ± 9.2	349 ± 8.0
DR-1	7.89	26.72	26.39	222	247	284
DR-2	8.06	27.39	28.22	232	284	320
DR-3	7.50	31.22	32.28	214	267	314
DR-4	7.33	28.50	27.78	225	278	324
DR Ave	7.69 ± 0.34	28.46 ± 1.93*	28.67 ± 2.53**	223 ± 7.5	269 ± 16.3	311 ± 18.0***

CR: Control Rat; DR: Diabetic Rat; Ave: average; Data are expressed as the mean ± standard deviation from 4 animals. * p = 0.0003, vs. CR. ** p = 0.0004, vs. CR. *** p = 0.0116, vs. CR (Student’s t-test).

Effect of Diabetes on the Expression of RTKs and Changes in Arterial Tissue Structure

Immunohistochemistry was used to evaluate the expression levels of DDR2, DDR1, EGFR, and α-smooth muscle actin (α-SMA) in the arterial tissues of non-diabetic and diabetic rats (Fig. 4A). Both DDR2 and DDR1 were expressed at significantly elevated levels in the underlying medial SMCs of arterial tissue of diabetic rats compared to non-diabetic rats with increasing 63.14 and 75.51%, respectively. In contrast, the expression levels of EGFR and α-SMA in arterial tissues were not different (EGFR) or slightly increased (α-SMA) between non-diabetic and diabetic rats (Fig. 4B, Supplementary Table S2). These results suggest that the elevations in protein expression in SMCs of arterial tissue due to diabetes might be specific to the DDR family among the RTKs.

Fig. 4. Expression Levels of DDR2, DDR1, EGFR, and α-SMA in Arterial Tissues of Control and Diabetic Rats

(A) Immunohistochemical analysis was performed on serial sections of aortae from control and diabetic groups. Sections were stained with antibodies recognizing DDR2, DDR1, EGFR, and α-SMA, and indicated by arrows. Images were captured at 400× magnification. (B) The amount of DDR2, DDR1, EGFR, and α-SMA in arterial tissues of control and diabetic rats was quantified by the integrated density or equal to MEAN gray values × pixel number in the ImageJ software. Each column with a vertical bar represents the mean ± standard deviation from 4 animals. * p = 0.0190, vs. control rat. ** p = 0.0124, vs. control rat (Student’s t-test).

DISCUSSION

RTK family proteins play pivotal roles in controlling cell proliferation, growth arrest, and differentiation by posttranslational processes including folding, covalent modification, and trafficking from the ER to the cell membrane via the Golgi apparatus. Because they must be present at the cell surface to function, any mechanisms controlling their presentation on the cell surface are important for their activity.⁴⁰⁾ For DDR proteins to become functional molecules, following their synthesis in the ER, they must translocate to the cell surface, where they transmit extracellular signals to the inside of the cell. When the glucose in the culture medium is depleted, the DDRs develop immature N-glycosyl carbohydrates, and the proteins become trapped within the ER, as the regulatory mechanism by which DDRs are expressed at the cell surface is dependent on glucose.⁸⁾ In this study, at least four isoforms of DDR2 and three isoforms of DDR1 with distinct molecular masses were detected when the DDR2 and DDR1 proteins were expressed in HEK293 cells. Of these isoforms, we found that only the highest molecular mass forms, with molecular weights of 130 kDa for DDR2 and 120 kDa for DDR1, were functional forms that could interact with collagen in the extracellular matrix to mediate tyrosine auto-phosphorylation. By using tunicamycin (inhibitor of N-glycosylation of glucose transporter-1) and alloxan (O-glycosylation inhibitor) treatment, we figured out that the active DDRs were generated by the N-glycosylation process.⁸⁾ However, EGFR presented only one isoform independent of the glucose concentration in the culture medium. We have also experimented to evaluate the active form of DDR2 from the detached cells and extracellular matrix from HEK293-DDR2 cells cultured in collagen-coated wells. The results showed that only the active form of DDR2 in the extracellular matrix fraction was tyrosine phosphorylated.⁸⁾

We generally detected multiple isoforms of DDR family proteins in transfection experiments involving cultures of HEK293 cells, primary aortic vascular SMCs, and cancer cell lines such as HT1080 and MDA-MB-231. In contrast, EGFR, as an example of another RTK, was detected mainly at a single molecular weight. After testing the generation of multiple isoforms of DDR protein under the glucose concentration from zero to 20 mmol/L. In the remaining high glucose concentration, the active forms of DDRs remained the main form. In contrast, when the glucose concentration was depleted, the incomplete forms of non-glycosylated DDRs remained at a high level; and the lowest molecular weight form of EGFR was hardly detectable even after prolonged culture in the depleted glucose medium. These results suggest that DDR family proteins are stable and remain in incomplete or complete isoforms depending on glucose concentration in the environment. In the low glucose concentration, the incomplete form is the majority and when the glucose supply is restored, the translocation of DDRs to the cell surface from the ER is induced. To date, there has been no research to show the low-glucose promoted protein degradation of cell surface N-glycosylated DDRs. Based on our results, we suggest the hypothesis that in the low glucose concentration, the DDRs remained in ER under incomplete forms and their translocation to the cell surface quickly under active forms when glucose is supplied.

The regulation of DDRs by glucose might have pathological significance in various diabetic complications, such as atherosclerosis and nephropathy, and could be associated with the development of clinical conditions.^20–22) DDRs have also been implicated in human cancers and metastasis, and a high level of hexosamine is required for them to be present at high levels at the cell surface in functional form.^{23–25,41,42)} For animal models, streptozotocin-induced diabetic rats were used widely with streptozotocin concentration from 50 to 100 mg/kg.^43–45) We also used streptozotocin combined with nicotinamide to develop the type 2 diabetic rats for our experiments. In this study, the results on transgenic HEK293 cell lines expressing DDRs, primary cells, and two cancer cell lines (HT1080 and MDA-MB-132) indicate that under conditions of high glucose concentrations, the active form of DDRs is expressed mainly on the cell surface. The collagen I can only attached to the active form of DDRs. Therefore, under high glucose concentration conditions, the expression levels of DDR1 and DDR2 increased by 75.51 and 63.14%, respectively. Increased expression of the active DDRs leads to increased expression of fibronectin and collagen type I in extracellular fibroblasts which is a binding molecule in the process of arterial fibrosis. These results are consistent with the previous research results of Titus et al.³⁴⁾ Also in this study, the authors used metformin—a substance widely used in anti-diabetic treatment to test these receptors’ suppression. Interestingly, the results have shown that metformin also inhibits the expression of DDR2, thereby inhibiting the expression of fibronectin and collagen type 1. Since DDR2 is considered a target molecule of metformin and it plays an important role in fibroblasts and may be directly involved in diabetic cardiomyopathy.³⁴⁾

In summary, our results suggest that glucose regulates the cellular activity of DDRs by controlling their trafficking to the cell membrane. Interestingly, the activity of DDRs is controlled by glucose, an essential nutrient for metabolism in the body and important for maintaining homeostatic balance. At this time, it has been evident at the molecular level that high glucose promoted the high expression of fibronectin and collagen type 1 via increasing the active DDR2 in diabetic animals. The data generated in this study provide initial knowledge for further discovering the molecular mechanisms of this process in the future.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol. Cell, 1, 13–23 (1997).
• 2) Leitinger B. Transmembrane collagen receptors. Annu. Rev. Cell Dev. Biol., 27, 265–290 (2011).
• 3) Fu HL, Valiathan RR, Arkwright R, Sohail A, Mihai C, Kumarasiri M, Mahasenan KV, Mobashery S, Huang P, Agarwal G, Fridman R. Discoidin domain receptors: unique receptor tyrosine kinases in collagen mediated signaling. J. Biol. Chem., 288, 7430–7437 (2013).
• 4) Carafoli F, Mayer MC, Shiraishi K, Pecheva MA, Chan LY, Nan R, Leitinger B, Hohenester E. Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling. Structure, 20, 688–697 (2012).
• 5) Curat CA, Eck M, Dervillez X, Vogel WF. Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding. J. Biol. Chem., 276, 45952–45958 (2001).
• 6) Leitinger B. Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2. J. Biol. Chem., 278, 16761–16769 (2003).
• 7) Phan TN, Wong EL, Park SY, Kim HJ, Yang BS. Defective Ca²⁺ binding in a conserved binding site causes incomplete N-glycan processing and endoplasmic reticulum trapping of discoidin domain receptors. Biosci. Biotechnol. Biochem., 79, 574–580 (2015).
• 8) Phan TN, Wong EL, Sun X, Kim G, Jung SH, Yoon CN, Yang BS. Low stability and a conserved N-glycosylation site are associated with regulation of the discoidin domain receptor family by glucose via post-translational N-glycosylation. Biosci. Biotechnol. Biochem., 77, 1907–1916 (2013).
• 9) Ali BR, Xu H, Akawi NA, John A, Karuvantevida NS, Langer R, Al-Gazali L, Leitinger B. Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients. Hum. Mol. Genet., 19, 2239–2250 (2010).
• 10) Al-Kindi A, Kizhakkedath P, Xu H, John A, Sayegh AA, Ganesh A, Al-Awadi M, Al-Anbouri L, Al-Gazali L, Leitinger B, Ali BR. A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intracellular trafficking. BMC Med. Genet., 15, 42 (2014).
• 11) Ruiz PA, Jarai G. Discoidin domain receptors regulate the migration of primary human lung fibroblasts through collagen matrices. Fibrogenesis Tissue Repair, 5, 3 (2012).
• 12) Huang TL, Mu N, Gu JT, Shu Z, Zhang K, Zhao JK, Zhang C, Hao Q, Li WN, Zhang WQ, Liu NN, Zhang Y, Zhang W, Xue XC, Zhang YQ. DDR2-CYR61-MMP1 signaling pathway promotes bone erosion in rheumatoid arthritis through regulating migration and invasion of fibroblast-like synoviocytes. J. Bone Miner. Res., 32, 407–418 (2017).
• 13) Curat CA, Vogel WF. Discoidin domain receptor 1 controls growth and adhesion of mesangial cells. J. Am. Soc. Nephrol., 13, 2648–2656 (2002).
• 14) Olaso E, Labrador JP, Wang L, Ikeda K, Eng FJ, Klein R, Lovett DH, Lin HC, Friedman SL. Discoidin domain receptor 2 regulates fibroblast proliferation and migration through the extracellular matrix in association with transcriptional activation of matrix metalloproteinase-2. J. Biol. Chem., 277, 3606–3613 (2002).
• 15) Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR. Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1. J. Cell Biol., 180, 1277–1289 (2008).
• 16) Walsh LA, Nawshad A, Medici D. Discoidin domain receptor 2 is a critical regulator of epithelial-mesenchymal transition. Matrix Biol., 30, 243–247 (2011).
• 17) Labrador JP, Azcoitia V, Tuckermann J, Lin C, Olaso E, Mañes S, Brückner K, Goergen JL, Lemke G, Yancopoulos G, Angel P, Martínez C, Klein R. The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism. EMBO Rep., 2, 446–452 (2001).
• 18) Kano K, Marín de Evsikova C, Young J, Wnek C, Maddatu TP, Nishina PM, Naggert JK. A novel dwarfism with gonadal dysfunction due to loss-of-function allele of the collagen receptor gene, Ddr2, in the mouse. Mol. Endocrinol., 22, 1866–1880 (2008).
• 19) Bargal R, Cormier-Daire V, Ben-Neriah Z, Le Merrer M, Sosna J, Melki J, Zangen DH, Smithson SF, Borochowitz Z, Belostotsky R, Raas-Rothschild A. Mutations in DDR2 gene cause SMED with short limbs and abnormal calcifications. Am. J. Hum. Genet., 84, 80–84 (2009).
• 20) Ferri N, Carragher NO, Raines EW. Role of discoidin domain receptors 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in atherosclerosis and lymphangioleiomyomatosis. Am. J. Pathol., 164, 1575–1585 (2004).
• 21) Flamant M, Placier S, Rodenas A, Curat CA, Vogel WF, Chatziantoniou C, Dussaule JC. Discoidin domain receptor 1 null mice are protected against hypertension-induced renal disease. J. Am. Soc. Nephrol., 17, 3374–3381 (2006).
• 22) Guerrot D, Kerroch M, Placier S, Vandermeersch S, Trivin C, Mael-Ainin M, Chatziantoniou C, Dussaule JC. Discoidin domain receptor 1 is a major mediator of inflammation and fibrosis in obstructive nephropathy. Am. J. Pathol., 179, 83–91 (2011).
• 23) Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell, 131, 1190–1203 (2007).
• 24) Hammerman PS, Sos ML, Ramos AH, et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov., 1, 78–89 (2011).
• 25) Huang Z, Yao H, Yang Z. Prognostic significance of TM4SF1 and DDR1 expression in epithelial ovarian cancer. Oncol. Lett., 26, 448 (2023).
• 26) Liu J, Chiang HC, Xiong W, Laurent V, Griffiths SC, Dülfer J, Deng H, Sun X, Yin YW, Li W, Audoly LP, An Z, Schürpf T, Li R, Zhang N. A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer. J. Immunother. Cancer, 11, e006720 (2023).
• 27) Wang X, Liu C. miR-199a-3p promotes gastric cancer progression by promoting its stemness potential via DDR2 mediation. Cell. Signal., 106, 110636 (2023).
• 28) Hou G, Vogel WF, Bendeck MP. The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. J. Clin. Invest., 107, 727–735 (2001).
• 29) Hou G, Vogel WF, Bendeck MP. Tyrosine kinase activity of discoidin domain receptor 1 is necessary for smooth muscle cell migration and matrix metalloproteinase expression. Circ. Res., 90, 1147–1149 (2002).
• 30) Khoo CL, Perera M. Diabetes and the menopause. J. Br. Menopause Soc., 11, 6–11 (2005).
• 31) Lincoln TM, Dey N, Sellak H. cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J. Appl. Physiol., 91, 1421–1430 (2001).
• 32) Kim SW, Jeong SJ, Munarriz R, Kim NN, Goldstein I, Traish AM. Role of the nitric oxide-cyclic GMP pathway in regulation of vaginal blood flow. Int. J. Impot. Res., 15, 355–361 (2003).
• 33) Kim NN, Christianson DW, Traish AM. Role of arginase in the male and female sexual arousal response. J. Nutr., 134 (Suppl.), 2873S–2879S, discussion, 2895S (2004).
• 34) Titus AS, Ushakumary MG, Venugopal H, Wang M, Lakatta EG, Kailasam S. Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in adventitial fibroblasts via inhibition of discoidin domain receptor 2. Int. J. Mol. Sci., 24, 585 (2022).
• 35) Siddiqui K, Kim GW, Lee DH, Shin HR, Yang EG, Lee NT, Yang BS. Actinomycin D identified as an inhibitor of discoidin domain receptor 2 interaction with collagen through an insect cell based screening of a drug compound library. Biol. Pharm. Bull., 32, 136–141 (2009).
• 36) Orlandi A, Ehrlich HP, Ropraz P, Spagnoli LG, Gabbiani G. Rat aortic smooth muscle cells isolated from different layers and at different times after endothelial denudation show distinct biological features in vitro. Arterioscler. Thromb., 14, 982–989 (1994).
• 37) Furman BL. Streptozotocin-induced diabetic models in mice and rats. Curr. Protoc., 1, e78 (2021).
• 38) Sun X, Kim YH, Phan TN, Yang BS. Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population. Biosci. Biotechnol. Biochem., 78, 1805–1812 (2014).
• 39) Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods, 9, 671–675 (2012).
• 40) Molinari M. N-glycan structure dictates extension of protein folding or onset of disposal. Nat. Chem. Biol., 3, 313–320 (2007).
• 41) Badiola I, Villacé P, Basaldua I, Olaso E. Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability. Oncol. Rep., 26, 971–978 (2011).
• 42) Valencia K, Ormazábal C, Zandueta C, Luis-Ravelo D, Antón I, Pajares MJ, Agorreta J, Montuenga LM, Martínez-Canarias S, Leitinger B, Lecanda F. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces cell survival, homing, and colonization in lung cancer bone metastasis. Clin. Cancer Res., 18, 969–980 (2012).
• 43) Higashi Y, Higashi K, Mori A, Sakamoto K, Ishii K, Nakahara T. Anti-cataract effect of resveratrol in high-glucose-treated streptozotocin-induced diabetic rats. Biol. Pharm. Bull., 41, 1586–1592 (2018).
• 44) Otake H, Mano Y, Deguchi S, Ogata F, Kawasaki N, Nagai N. Effect of ointment base on the skin wound-healing deficits in streptozotocin-induced diabetic rat. Biol. Pharm. Bull., 46, 707–712 (2023).
• 45) Noda M, Kikuchi C, Tarui R, Nakamura T, Murase T, Hori E, Matsunaga T. Effect of topiroxostat on reducing oxidative stress in the aorta of streptozotocin-induced diabetic rats. Biol. Pharm. Bull., 46, 272–278 (2023).