TLR7 (Toll-Like Receptor 7) Facilitates Heme Scavenging Through the BTK (Bruton Tyrosine Kinase)-CRT (Calreticulin)-LRP1 (Low-Density Lipoprotein Receptor-Related Protein-1)-Hx (Hemopexin) Pathway in Murine Intracerebral Hemorrhage.

Background and Purpose- Heme and iron are considered to be key factors responsible for secondary insults after intracerebral hemorrhage (ICH). Our previous study showed that LRP1 (low-density lipoprotein receptor-related protein-1)-Hx (hemopexin) facilitates removal of heme. The TLR7 (Toll-like receptor 7)-BTK (Bruton tyrosine kinase)-CRT (calreticulin) pathway regulates the expression of LRP1-Hx. This study is designed to clarify whether TLR7 activation facilitates heme scavenging and to establish the potential role of the BTK-CRT-LRP1-Hx signaling pathway in the pathophysiology of ICH. Methods- ICH was induced by stereotactic, intrastriatal injection of type VII collagenase. Mice received TLR7 agonist (imiquimod) via intraperitoneal injection after ICH induction. TLR7 inhibitor (ODN2088), BTK inhibitor (LFM-A13), and CRT agonist (thapsigargin) were given in different groups to further evaluate the underlying pathway. Mice were randomly divided into sham, ICH+vehicle (normal saline), ICH+Imiquimod (2.5, 5, and 10 µg/g), ICH+ODN2088, ICH+LFM-A13, ICH+thapsigargin, and ICH+ODN2088+thapsigargin. Imiquimod was administered twice daily starting at 6 hours after ICH; ODN2088 was administered by intracerebroventricular injection at 30 minutes, and LFM-A13 or thapsigargin was administered by intraperitoneal injection at 3 hours after ICH induction. Neurological scores, cognitive abilities, as well as brain edema, blood-brain barrier permeability, hemoglobin level, brain expression of TLR7/BTK/CRT/LRP1/Hx were analyzed. Results- Low dosage imiquimod significantly attenuated hematoma volume, brain edema, BBB permeability, and neurological deficits after ICH. Imiquimod also increased protein expressions of TLR7, BTK, CRT, LRP1, and Hx; ODN2088 reduced TLR7, BTK, CRT, LRP1, and Hx expressions. Conclusions- TLR7 plays an important role in heme scavenging after ICH by modulating the BTK-CRT-LRP1-Hx pathway. TLR7 may offer protective effects by promoting heme resolution and reduction of brain edema after ICH.

Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.

Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N-terminal domain of TSP1 binds to the calreticulin-LRP1 receptor co-complex to signal down-regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin-LRP1 activates cell survival signals such as PI3-kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion-independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N-terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin-LRP1 binding site [amino acids 17-35 (hep I)] in the N-terminal domain promotes fibroblast survival under anchorage-independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1-mediated anchorage-independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage-independent survival. These data define a novel role for TSP1 signaling through the calreticulin/LRP1 co-complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.-Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy-Ullrich, J. E. Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.