Many research groups have reported that AGEs induce proinflammatory cytokine production through the stimulation of RAGE receptors, resulting in inflammatory diseases [3]. These signaling cascades are regarded as factors that exacerbate various lifestyle- and age-related diseases, particularly diabetic complications [20]. However, the pathological consequences and physiological functions of AGEs have not yet been characterized in detail due to their structural heterogeneity and the existence of various receptors other than RAGE.
Among AGEs, Glycol-AGEs have been reported to suppress mitochondrial functions in Achilles tendon-derived fibroblasts [21] and induce apoptosis in osteoblasts by endoplasmic reticulum stress [22]. Furthermore, the intraperitoneal administration of Glycol-AGEs to rats induced the functional and structural remodeling of cardiomyocytes, resulting in cardiac dysfunction [23].
In the present study, Glycol-AGEs, which are representative toxic AGEs, promoted the proliferation of RAW264.7 cells via the JAK-STAT pathway. This effect was biphasic, showing a concentration-dependent increase at a low concentration range of 0–10 µg/mL and attenuation at 100 µg/mL. TNF-α was produced in cells exposed to 100 µg/mL Glycol-AGEs, which induced an inflammatory state via the autocrine effect. Although TNF-α induces cell death, including apoptosis [24], the LDH release assay revealed no cytotoxicity by Glycol-AGEs at 100 µg/mL. These results suggest that no relationship exists between TNF-α production and the attenuation of cell proliferation by a stimulation with Glycol-AGEs at 100 µg/mL. A previous study reported that Tob1, a modulator of the expression and activities of cell cycle proteins, such as CDKs, inhibited the proliferation of hepatocytes under normal conditions; however, its expression was down-regulated by hepatectomy and returned to its original level after cell regeneration, resulting in the biphasic regulation of cell proliferation by a change in Tob1 expression [25]. Therefore, although the biphasic effects of the promotion of macrophage proliferation in this study may be regulated by an unknown modulator, such as Tob1, further studies are warranted to clarify this issue. Since AGEs are constantly present in healthy tissues that do not show a significant inflammatory response, the promotion of macrophage proliferation by Glycol-AGEs may play a physiological role in maintaining or boosting innate immune responses or may function as a presymptomatic risk factor for inflammatory diseases, such as hyperplasia and fibrosis.
We previously established triple receptor KO RAW264.7 cells lacking RAGE, TLR4, and TLR2 to examine AGEs-induced inflammatory responses. These triple KO cells did not produce TNF-α, even when stimulated with glyceraldehyde-derived AGEs at 500 µg/mL [9]. To elucidate the involvement of inflammatory-related receptors (RAGE-TLR4-TLR2) in the Glycol-AGEs-induced promotion of cell proliferation, the proliferation of KO cells stimulated with Glycol-AGEs was compared with that of wild-type cells. The obtained results showed that Glycol-AGEs promoted the proliferation of KO and wild-type cells to the same extent, and indicated that cell proliferation was induced by Glycol-AGEs mediated by a receptor pathway other than RAGE, TLR4, and TLR2. A number of non-inflammatory AGEs receptors, such as CD36, FEEL, LOX-1, SR-A, and SR-BI, have been identified; however, they were shown to function as scavenger receptors for AGEs but not as the cell proliferative receptors [26, 27, 28, 29]. The receptors responsible for Glycol-AGEs-induced cell proliferation remain unknown, and the significance of the present results has yet to be established.
We found that the JAK2/STAT5 signaling pathway was important in the Glycol-AGEs-induced promotion of cell proliferation. Additionally, its proliferative effects were accompanied by the up-regulated expression of some cell-cycle genes, which plateaued at 10 µg/mL. The JAK2/STAT5 signaling pathway was previously shown to contribute to the production of GM-CSF in microglia [30] and MCP-1 in the monocytic cell line U937 [31] in their activated states. Furthermore, it has been associated with erythropoietin receptors, prolactin receptors [32, 33], and some cytokine receptors for IL-2, 3, and 5 [34, 35, 36], suggesting its involvement in the proliferation of some cells, including fibroblasts and immune cells. Therefore, JAK/STAT appears to play an important role in the Glycol-AGEs-induced promotion of cell proliferation.
The present results revealed that Glycol-AGEs promoted the proliferation of macrophages via the JAK2/STAT5 pathway at concentrations that were incapable of inducing the production of TNF-α. These results suggest that Glycol-AGEs in vivo contribute not only to the exacerbation of pathological conditions by inducing excessive inflammatory responses, but also to the maintenance and activation of innate immunity, including macrophage proliferation. Furthermore, these findings may present a novel physiological potential of glycation as the post-transcriptional modifications. The significance of the Glycol-AGEs-induced promotion of cell proliferation remains unclear and, thus, further studies are warranted.