Cyclin D3 Mediates Synthesis of a Hyaluronan Matrix That Is Adhesive for Monocytes in Mesangial Cells Stimulated to Divide in Hyperglycemic Medium

Serum-starved, growth-arrested, near confluent rat mesangial cell cultures were stimulated to divide in medium with low (5.6 mm) or high (25.6 mm) glucose. In high glucose cultures Western blots showed large increases in cyclin D3 and CCAAT/enhancer-binding protein α (C/EBPα) at 48–72 h, concurrent with the production of a monocyte-adhesive hyaluronan matrix, whereas low glucose and mannitol osmotic control cultures did not. Cyclin D3 small interfering RNA inhibited both the synthesis of this matrix and the up-regulation of C/EBPα in cultures exposed to high glucose, indicating that cyclin D3 is a key mediator in regulating responses of dividing mesangial cells to hyperglycemia. A complex with cyclin D3, cyclin-dependent kinase 4, and C/EBPα was observed at 48–72 h in the hyperglycemic cultures, and cyclin D3 and C/EBPα were spatially co-localized in coalesced perinuclear honeycomb-like structures with embedded hyaluronan. Furthermore, microtubule-associated protein 1 light chain 3, a marker for autophagy, colocalizes with these structures. These results suggest that cyclin D3 is a central coordinator that controls the organization of a complex set of proteins that regulate autophagy, formation of the monocyte-adhesive hyaluronan matrix, and C/EBPα-mediated lipogenesis. Abnormal deposits of hyaluronan, cyclin D3, and C/EBPα were present in glomeruli of kidney sections from hyperglycemic rats 4 weeks after streptozotocin treatment, indicating that similar processes likely occur in vivo. Mesangial cell cultures treated with poly(I:C) or tunicamycin in normal glucose media synthesized monocyte-adhesive hyaluronan matrices but with concurrent down-regulation of cyclin D3. This indicates that the cyclin D3 mechanism is induced by hyperglycemia and is distinct from those involved in these cell stress responses.One of the abnormalities detected after the onset of hyperglycemic diabetes in the streptozotocin rat model is an early (already by 3 days) and self-limited proliferation of glomerular mesangial cells that is associated with de novo expression of α-smooth muscle actin, an activation marker of the proliferative mesangial cell phenotype (1–3). After this early transient proliferation and phenotypic activation, there is a prominent glomerular infiltration of monocytes and macrophages (3). Our previous study showed that abnormal hyaluronan matrices also form in the hyperglycemic glomeruli within 1 week (4). We also showed that quiescent, growth-arrested rat mesangial cells, stimulated to divide in a hyperglycemic level of glucose (25.6 mm), form a hyaluronan matrix that is adhesive for U937 monocytic cells. These results suggest that there is an important link in vivo between mesangial cell division in response to hyperglycemia, glomerular hyaluronan matrix synthesis, and the accumulation of monocytes/macrophages in glomerular diabetic nephritis.Previous studies have shown that smooth muscle cell cultures exposed to tunicamycin (endoplasmic reticulum stress) or poly(I:C) (viral mimetic) synthesize hyaluronan cable-like structures that are adhesive for monocytes (5, 6). The experiments described in this report indicate that growth-arrested mesangial cells stimulated to divide in hyperglycemic medium synthesize similar structures by a distinctly different mechanism that requires protein kinase C up-regulation at the initiation of cell division and subsequent up-regulation of cyclin D3 after completion of cell division. The up-regulation of cyclin D3 in turn appears to control an autophagic response and is coordinate with up-regulation of C/EBPα, a factor that controls lipogenic responses. Evidence is also provided that cyclin D3 and C/EBPα also contribute to glomerular responses to hyperglycemia in vivo.     

Heparin Prevents Intracellular Hyaluronan Synthesis and Autophagy Responses in Hyperglycemic Dividing Mesangial Cells and Activates Synthesis of an Extensive Extracellular Monocyte-adhesive Hyaluronan Matrix after Completing Cell Division

Growth-arrested rat mesangial cells (RMCs) at a G₀/G₁ interphase stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy/cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division. This study shows that heparin inhibits the intracellular hyaluronan synthesis and autophagy responses, but at the end of cell division it induces synthesis of a much larger extracellular monocyte-adhesive hyaluronan matrix. Heparin bound to RMC surfaces by 1 h, internalizes into the Golgi/endoplasmic reticulum region by 2 h, and was nearly gone by 4 h. Treatment by heparin for only the first 4 h was sufficient for its function. Streptozotocin diabetic rats treated daily with heparin showed similar results. Glomeruli in sections of diabetic kidneys showed extensive accumulation of autophagic RMCs, increased hyaluronan matrix, and influx of macrophages over 6 weeks. Hyaluronan staining in the glomeruli of heparin-treated diabetic rats was very high at week 1 and decreased to near control level by 6 weeks without any RMC autophagy. However, the influx of macrophages by 6 weeks was as pronounced as in diabetic glomeruli. The results are as follows: 1) heparin blocks synthesis of hyaluronan in intracellular compartments, which prevents the autophagy and cyclin D3 responses thereby allowing RMCs to complete cell division and sustain function; 2) interaction of heparin with RMCs in early G₁ phase is sufficient to induce signaling pathway(s) for its functions; and 3) influxed macrophages effectively remove the hyaluronan matrix without inducing pro-fibrotic responses that lead to nephropathy and proteinurea in diabetic kidneys.     

The Responses of Hyperglycemic Dividing Mesangial Cells to Heparin Are Mediated by the Non-reducing Terminal Trisaccharide

Our previous studies showed: (i) that growth-arrested G₀/G₁ rat mesangial cells stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy and the cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division; and (ii) that heparin inhibits the intracellular hyaluronan and autophagy responses, but after completing division, induces hyaluronan synthesis at the plasma membrane with the formation of a larger monocyte-adhesive hyaluronan matrix. This study shows: (i) that the non-terminal trisaccharide of heparin is sufficient to initiate the same responses as intact heparin, (ii) that a fully sulfated tetrasaccharide isolated from bacterial heparin lyase 1 digests of heparin that contains a Δ-2S-iduronate on the non-reducing end does not initiate the same responses as intact heparin, and (iii) that removal of the Δ-2S-iduronate to expose the fully sulfated trisaccharide (GlcNS(6S)-IdoUA(2S)-GlcNS(6S)) does initiate the same responses as intact heparin. These results provide evidence that mammalian heparanase digestion of heparin and heparan sulfate exposes a cryptic motif on the non-reducing termini that is recognized by a receptor on dividing cells.     

The Expression of Cyclin D1 during Adipogenesis in Pig Primary Stromal–Vascular Cultures

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal‐vascular (S‐V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S‐V cells using 8% sodium dodecyl sulfate‐polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti‐mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti‐CCAAT/enhancer binding protein (C/EBPα) and anti‐cyclin D1 together. Cyclin D1 expression was evaluated in 105‐day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S‐V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid‐filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.     

Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α) in human colon cancer cells

To explore the relationship between UPR and autophagy in intestinal epithelial cells, we investigated whether autophagy was induced by endoplasmic reticulum (ER) stress in colon cancer cell lines. We demonstrated that autophagy was induced by ER stress in HT29, SW480, and Caco-2 cells. In these cells, inositol-requiring enzyme1α (IRE1α) and C/EBP homologous protein (CHOP) were involved in the ER stress–autophagy pathway, and CHOP was a regulator of IRE1α protein expression. Our findings suggest that CHOP promotes IRE1α and autophagy especially in ER stress conditions. This study will provide important insights into the disclosure of the ER stress–autophagy pathway.     

Hyaluronan matrices in pathobiological processes

Hyaluronan matrices are ubiquitous in normal and pathological biological processes. This remarkable diversity is related to their unique mechanism of synthesis by hyaluronan synthases. These enzymes are normally activated in the plasma membrane and utilize cytosolic substrates directly to form these large polyanionic glycosaminoglycans, which are extruded directly into the extracellular space. The extracellular matrices that are formed interact with cell surface receptors, notably CD44, that often dictate the biological processes, as described in the accompanying minireviews of this series. This article focuses on the discovery in recent studies that many cell stress responses initiate the synthesis of a monocyte-adhesive hyaluronan extracellular matrix, which forms a central focus for subsequent inflammatory processes that are modulated by the dialogue between the matrix and the inflammatory cells. The mechanisms involve active hyaluronan synthases at the cell membrane when cell stresses occur at physiological levels of glucose. However, dividing cells at hyperglycemic levels of glucose initiate the synthesis of hyaluronan in intracellular compartments, which induces endoplasmic reticulum stress and autophagy, processes that probably contribute greatly to diabetic pathologies.     

Mycobacterial lipoprotein activates autophagy via TLR2/1/CD14 and a functional vitamin D receptor signalling

In human monocytes, Toll‐like receptor (TLR) 2/1 activation leads to vitamin D3‐dependent antimycobacterial activities, but the molecular mechanisms by which TLR2/1 stimulation induces antimicrobial activities against mycobacteria remain unclear. Here we show that TLR2/1/CD14 stimulation by mycobacterial lipoprotein LpqH can robustly activate antibacterial autophagy through vitamin D receptor signalling activation and cathelicidin induction. We found that CCAAT/enhancer‐binding protein (C/EBP)‐β‐dependent induction of 25‐hydroxycholecalciferol‐1α‐hydroxylase (Cyp27b1) hydroxylase was critical for LpqH‐induced cathelicidin expression and autophagy. In addition, increases in intracellular calcium following AMP‐activated protein kinase (AMPK) activation played a crucial role in LpqH‐induced autophagy. Moreover, AMPK‐dependent p38 mitogen‐activated protein kinase (MAPK) activation was required for LpqH‐induced Cyp27b1 expression and autophagy activation. Collectively, these data suggest that TLR2/1/CD14‐Ca²⁺‐AMPK‐p38 MAPK pathways contribute to C/EBP‐β‐dependent expression of Cyp27b1 and cathelicidin, which played an essential role in LpqH‐induced autophagy. Furthermore, these results establish a previously uncharacterized signalling pathway of antimycobacterial host defence through a functional link of TLR2/1/CD14‐dependent sensing to the induction of autophagy.     

Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade

Adult mice abundantly express neudesin, an extracellular heme-binding protein with neurotrophic activity, in white adipose tissues. At the early stage of adipocyte differentiation during adipogenesis, however, the expression of neudesin decreased transiently. Neudesin-hemin significantly suppressed adipogenesis in 3T3-L1 cells. The knockdown of neudesin by RNA interference markedly promoted adipogenesis in 3T3-L1 cells and decreased MAPK activation during adipocyte differentiation. The addition or knockdown of neudesin affected the expression of C/EBPα and PPARγ but not of C/EBPβ. These findings suggest that neudesin plays a critical role in the early stage of adipocyte differentiation in which C/EBPβ induces PPARγ and C/EBPα expressions, by controlling the MAPK pathway.     

Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion

Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms.     

Cells lacking IKKα show nuclear cyclin D1 overexpression and a neoplastic phenotype: role of IKKα as a tumor suppressor

The catalytic subunits of IκB kinase (IKK) complex, IKKα and IKKβ, are involved in activation of NF-κB and in mediating a variety of other biological functions. Though these proteins have a high-sequence homology, IKKα exhibits different functional characteristics as compared with IKKβ. Earlier, we have shown that cyclin D1 is overexpressed and predominantly localized in the nucleus of IKKα(-/-) cells, indicating that IKKα regulates turnover and subcellular distribution of cyclin D1, which is mediated by IKKα-induced phosphorylation of cyclin D1. Because cyclin D nuclear localization is implicated in tumor development, we examined whether the absence of IKKα leads to tumor development as well. In the current study, we show that IKKα plays a critical role in tumorigenesis. Though IKKα(-/-) MEF cells show a slower anchorage-dependent growth, they are clonogenic in soft agar. These cells are tumorigenic in nude mice. Microarray analysis of IKKα(-/-) cells indicates a differential expression of genes involved in proliferation and apoptosis. Furthermore, analysis of microarray data of human lung cancer cell lines revealed decreased IKKα RNA expression level as compared with cell lines derived from normal bronchial epithelium. These results suggest that IKKα may function as a tumor suppressor gene. Absence of IKKα may induce tumorigenicity by nuclear localization of cyclin D1 and modulating the expression of genes involved in neoplastic transformation.