The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus.

BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.

Integrin-mediated type II TGF-ß receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling.

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-ß mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-ß receptor (TßRII), which in turn promotes a TßRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TßRII is considered the principal regulatory mechanism of TßRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TßRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TßRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TßRII tail tyrosine residues, resulting in inhibition of TßR-dependent fibrotic signaling. The collagen-binding receptor integrin α1ß1 was required for recruitment of TCPTP to the TßRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TßRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1ß1 and TßRII that is essential for TßRII-mediated SMAD activation and fibrotic signaling pathways.

Receptor tyrosine kinases in the nucleus.

To date, 18 distinct receptor tyrosine kinases (RTKs) are reported to be trafficked from the cell surface to the nucleus in response to ligand binding or heterologous agonist exposure. In most cases, an intracellular domain (ICD) fragment of the receptor is generated at the cell surface and translocated to the nucleus, whereas for a few others the intact receptor is translocated to the nucleus. ICD fragments are generated by several mechanisms, including proteolysis, internal translation initiation, and messenger RNA (mRNA) splicing. The most prevalent mechanism is intramembrane cleavage by γ-secretase. In some cases, more than one mechanism has been reported for the nuclear localization of a specific RTK. The generation and use of RTK ICD fragments to directly communicate with the nucleus and influence gene expression parallels the production of ICD fragments by a number of non-RTK cell-surface molecules that also influence cell proliferation. This review will be focused on the individual RTKs and to a lesser extent on other growth-related cell-surface transmembrane proteins.

Regulated intramembrane cleavage of the EGF receptor.

Following the addition of EGF or ionomycin to A431 cells, protease activity mediates cleavage of the EGF receptor producing a 60 kDa fragment that includes the intracellular domain (ICD). This fragment is located in both membrane and nuclear fractions. On the basis of sensitivity to chemical inhibitors and overexpression of cDNAs, the rhomboid intramembrane proteases, not γ-secretase proteases, are identified as responsible for the cleavage event. Agonist-initiated cleavage occurs slowly over 3-24 h. Inhibition of calpain protease activity significantly increased the detectable level of ICD fragment.

Cetuximab/C225-induced intracellular trafficking of epidermal growth factor receptor.

The monoclonal antibody C225 interacts with the ectodomain of the epidermal growth factor (EGF) receptor (EGFR) to block ligand binding and initiates receptor endocytosis and intracellular trafficking. The data herein show that C225-dependent EGFR trafficking relocalizes the receptor to the endoplasmic reticulum (ER) and nucleus. This mechanism, which also involves interaction of the C225-internalized receptor with the Sec61 translocon within the ER, is, in most respects, analogous to the pathway previously described for EGF-induced trafficking to the ER and nucleus. However, although inhibition of receptor tyrosine kinase activity blocks EGF-induced nuclear localization of the receptor, the same kinase inhibitors stimulate C225-dependent nuclear localization of EGFR in the nucleus. In contrast, the kinase inhibitor Lapatinib fails to stimulate nuclear accumulation of the receptor in C225-treated cells and does not provoke receptor dimerization as do inhibitors that recognize the open conformation of the receptor kinase. This suggests that inhibitor-dependent receptor dimerization may facilitate C225-induced receptor trafficking.

Trafficking of receptor tyrosine kinases to the nucleus.

It has been known for at least 20 years that growth factors induce the internalization of cognate receptor tyrosine kinases (RTKs). The internalized receptors are then sorted to lysosomes or recycled to the cell surface. More recently, data have been published to indicate other intracellular destinations for the internalized RTKs. These include the nucleus, mitochondria, and cytoplasm. Also, it is recognized that trafficking to these novel destinations involves new biochemical mechanisms, such as proteolytic processing or interaction with translocons, and that these trafficking events have a function in signal transduction, implicating the receptor itself as a signaling element between the cell surface and the nucleus.

Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression.

The epidermal growth factor (EGF)-dependent trafficking of the intact EGF receptor to the nucleus and its requirement for growth factor induction of cyclin D and other genes has been reported. Unresolved is the mechanism by which this or other transmembrane proteins are excised from a lipid bilayer before nuclear translocalization. We report that, after the addition of EGF, the cell surface EGF receptor is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61beta, a component of the Sec61 translocon, and is retrotranslocated from the ER to the cytoplasm. Abrogation of Sec61beta expression prevents EGF-dependent localization of EGF receptors to the nucleus and expression of cyclin D. This indicates that EGF receptors are trafficked from the ER to the nucleus by a novel pathway that involves the Sec61 translocon.

Contrasting role of phospholipase C-gamma1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors.

While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-gamma1 (PLC-gamma1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-gamma1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-gamma1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-gamma1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-gamma1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-gamma1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-kappaB.

Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice.

Mice nullizygous for Plcg1 cease growing at early to mid-gestation. An examination of carefully preserved wild-type embryos shows clear evidence of erythropoiesis, but erythropoiesis is not evident in Plcg1 nullizygous embryos at the same stage. The analyses of embryonic materials demonstrate that in the absence of Plcg1, erythroid progenitors cannot be detected in the yolk sac or embryo body by three different assays, burst-forming units, colony-forming units, and analysis for the developmental marker Ter119. However, non-erythroid granulocyte/macrophage colonies are produced by Plcg1 null embryos. Further analysis of these embryos demonstrates significantly diminished vasculogenesis in Plcg1 nullizygous embryos based on the lack of expression of the endothelial marker platelet endothelial cell adhesion molecule-1. In addition, Plcg1 nullizygous embryos express a greatly reduced level of vascular endothelial growth factor receptor-2/Flk-1, consistent with significantly impaired vasculogenesis and erythropoiesis. Interestingly, these early embryos do express phospholipase C-gamma2, however, it is unable to substitute for the absence of phospholipase C-gamma1, which can be detected in its tyrosine-phosphorylated state.