Lactoferrin induces osteoblast proliferation and survival in vitro and is anabolic to bone in vivo. The molecular mechanisms by which lactoferrin exerts these biological actions are not known, but lactoferrin is known to bind to two members of the low-density lipoprotein receptor family, low- density lipoprotein receptor-related proteins 1 (LRP1) and 2 (LRP2). We have examined the role(s) of these receptors in the actions of lactoferrin on osteoblasts. We show that lactoferrin binds to cultured osteoblastic cells, and that LRP1 and LRP2 are expressed in several osteoblastic cell types. In primary rat osteoblastic cells, the LRP1/2 inhibitor receptor associated protein blocks endocytosis of lactoferrin and abrogates lactoferrin-induced p42/44 MAPK signaling and mitogenesis. Lactoferrin-induced mitogenesis is also inhibited by an antibody to LRP1. Lactoferrin also induces receptor associated protein-sensitive activation of p42/44 MAPK signaling and proliferation in osteoblastic human SaOS-2 cells, which express LRP1 but not LRP2. The mitogenic response of LRP1-null fibroblastic cells to lactoferrin is substantially reduced compared with that of cells expressing wild-type LRP1. The endocytic and signaling functions of LRP1 are independent of each other, because lactoferrin can activate mitogenic signaling in conditions in which endocytosis is inhibited. Taken together, these results 1) suggest that mitogenic signaling through LRP1 to p42/44 MAPKs contributes to the anabolic skeletal actions of lactoferrin; 2) demonstrate growth-promoting actions of a third LRP family member in osteoblasts; and 3) provide further evidence that LRP1 functions as a signaling receptor in addition to its recognized role in ligand endocytosis.

These results demonstrate that NaBu exerts an antihypertensive action, likely by suppressing the PRR-mediated intrarenal renin-angiotensin system.

The present study was designed to test the hypothesis that hypoxia inducible factor (HIF)-1α mediates profibrotic effects of angiotensin (ANG) II and to determine whether HIF prolyl-hydroxylase, the enzyme that promotes the degradation of HIF-1α, is involved in the profibrotic action of ANG II. In cultured renal medullary interstitial cells, ANG II (10−6 M) treatment for 20 hours remarkably increased HIF-1α levels, which was accompanied by the significant upregulation of collagen I/III and tissue inhibitor of metalloproteinases (TIMP)-1. HIF-1α siRNA decreased HIF-1α levels and completely blocked the effects of ANG II on collagen I/III and TIMP-1. HIF-1α siRNA also abolished ANG II-induced elevation of proliferating cell nuclear antigen, a marker of cell proliferation, and vimentin, a marker of cell transdifferentiation. HIF-2α siRNA did not affect the action of ANG II on collagen I/III and TIMP-1. Overexpression of PHD2 transgene, the predominant renal HIF prolyl-hydroxylase, attenuated ANG II-induced profibrotic action and silencing of PHD2 gene enhanced ANG II-induced profibrotic action. Removal of H2O2 eliminated ANG II-induced profibrotic effects. Two week ANG II infusion (150 ng/Kg/min) increased the expression of HIF-1α and α-smooth muscle actin in the renal medullary interstitial cells in vivo. Our data suggest that HIF-1α mediates ANG II-induced profibrotic effects through activation of cell transdifferentiation and that redox regulation of PHD plays a critical role in ANG II-induced activation of HIF-1α and consequent cell proliferation, transdifferentiation and abnormal extracellular matrix metabolism in renal cells.

Abstract-High salt induces the expression of transcription factor hypoxia-inducible factor (HIF) 1␣ and its target genes in the renal medulla, which is an important renal adaptive mechanism to high-salt intake. HIF prolyl-hydroxylase domain-containing proteins (PHDs) have been identified as major enzymes to promote the degradation of HIF-1␣. PHD2 is the predominant isoform of PHDs in the kidney and is primarily expressed in the renal medulla. The present study tested the hypothesis that PHD2 responds to high salt and mediates high-salt-induced increase in HIF-1␣ levels in the renal medulla. In normotensive rats, high-salt intake (4% NaCl, 10 days) significantly inhibited PHD2 expressions and enzyme activities in the renal medulla. Renal medullary overexpression of the PHD2 transgene significantly decreased HIF-1␣ levels. PHD2 transgene also blocked high-salt-induced activation of HIF-1␣ target genes heme oxygenase 1 and NO synthase 2 in the renal medulla. In Dahl salt-sensitive hypertensive rats, however, high-salt intake did not inhibit the expression and activities of PHD2 in the renal medulla. Correspondingly, renal medullary HIF-1␣ levels were not upregulated by high-salt intake in these rats. After transfection of PHD2 small hairpin RNA, HIF-1␣ and its target genes were significantly upregulated by high-salt intake in Dahl salt-sensitive rats. Overexpression of PHD2 transgene in the renal medulla impaired renal sodium excretion after salt loading. These data suggest that high-salt intake inhibits PHD2 in the renal medulla, thereby upregulating the HIF-1␣ expression. The lack of PHD-mediated response to high salt may represent a pathogenic mechanism producing salt-sensitive hypertension. (Hypertension. 2010;55:1129-1136.)

Sodium butyrate (NaBu) is reported to play important roles in a number of chronic diseases. The present work is aimed to investigate the effect of NaBu on angiotensin II (Ang II)‐induced cardiac hypertrophy and the underlying mechanism in in vivo and in vitro models. Sprague Dawley rats were infused with vehicle or Ang II (200 ng/kg/min) and orally administrated with or without NaBu (1 g/kg/d) for two weeks. Cardiac hypertrophy parameters and COX2/PGE2 pathway were analysed by real‐time PCR, ELISA, immunostaining and Western blot. The cardiomyocytes H9C2 cells were used as in vitro model to investigate the role of NaBu (2 mmol/L) in inhibition of Ang II‐induced cardiac hypertrophy. NaBu significantly attenuated Ang II‐induced increase in the mean arterial pressure. Ang II treatment remarkably increased cardiac hypertrophy as indicated by increased ratio of heart weight/body weight and enlarged cardiomyocyte size, extensive fibrosis and inflammation, as well as enhanced expression of hypertrophic markers, whereas hearts from NaBu‐treated rats exhibited a significant reduction in these hypertrophic responses. Mechanistically, NaBu inhibited the expression of COX2/PGE2 along with production of ANP and phosphorylated ERK (pERK) stimulated by Ang II in in vivo and in vitro, which was accompanied by the suppression of HDAC5 and HDAC6 activities. Additionally, knocking down the expression of HDAC5 and HDAC6 via gene‐editing strategy dramatically blocked Ang II‐induced hypertrophic responses through COX2/PGE2 pathway. These results provide solid evidence that NaBu attenuates Ang II‐induced cardiac hypertrophy by inhibiting the activation of COX2/PGE2 pathway in a HDAC5/HDAC6‐dependent manner.