Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity

Biomarker Potential of Vimentin in Oral Cancers

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker

Utility of Keratins as Biomarkers for Human Oral Precancer and Cancer

Human oral cancer is the single largest group of malignancies in the Indian subcontinent and the sixth largest group of malignancies worldwide. Squamous cell carcinomas (SCC) are the most common epithelial malignancy of the oral cavity, constituting over 90% of oral cancers. About 90% of OSCCs arise from pre-existing, potentially malignant lesions. According to WHO, OSCC has a 5-year survival rate of 45–60%. Late diagnosis, recurrence, and regional or lymph nodal metastases could be the main causes of the high mortality rates. Biomarkers may help categorize and predict premalignant lesions as high risk of developing malignancy, local recurrence, and lymph nodal metastasis. However, at present, there is a dearth of such markers, and this is an area of ongoing research. Keratins (K) or cytokeratins are a group of intermediate filament proteins that show paired and differentiation dependent expression. Our laboratory and others have shown consistent alterations in the expression patterns of keratins in both oral precancerous lesions and tumors. The correlation of these changes with clinicopathological parameters has also been demonstrated. Furthermore, the functional significance of aberrant keratins 8/18 expression in the malignant transformation and progression of oral tumors has also been documented. This article reviews the literature that emphasizes the value of keratins as biomarkers for the prognostication of human oral precancers and cancers

Biomarker Potential of Vimentin in Oral Cancers

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity

Vimentin knockdown phenotype was partially rescued upon re-expression of ΔNp63α in vimentin knockdown background.

<p>(A) Western blot analysis shows protein levels of ΔNp63 from whole cell lysates of vimentin knockdown and its vector control clones. (B) Fold change in mRNA expression level of ΔNp63 in vimentin knockdown as compared to vector control clones, using qRT-PCR analysis. (C) Western blot analysis shows protein levels of p21 and p27 from the whole cell lysates of vimentin knockdown and its vector control clones. (D) RT-PCR analysis shows expression of ΔNp63α, β and γ isoforms, between vimentin knockdown and vector control clones. GAPDH was used as a loading control. (E) Western blot analysis using anti-flag antibody confirmed the overexpression of flag-tagged ΔNp63α. (F) The protein level of vimentin was tested by western blotting in vimentin knockdown-vector control set and flag-ΔNp63α-vector control set, to confirm the maintenance of vimentin knockdown background in the second group. (G and H) qRT-PCR and western blot analysis of K5 and K14 in flag-ΔNp63α overexpressing and its vector control group. (I) Proliferation curves of flag-ΔNp63α overexpressing and its vector control clones over the period of 4 days, using MTT assay. (J) Representative image of clonogenic assay shows colonies formed by flag-ΔNp63α overexpressing and its vector control clones. (K) QRT-PCR analysis of differentiation specific markers K1, involucrin, filaggrin and loricrin. For all the qRT-PCR experiments, the relative expression of the target gene was normalized to the GAPDH. For all western blotting experiments, β-actin was used as a loading control. The graphical data represents ± SEM of three independent experiments.</p

Vimentin regulates differentiation switch via modulation of keratin 14 levels and their expression together correlates with poor prognosis in oral cancer patients

<div><p>Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchymal origin, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In view of the perceived role of keratins (Ks) as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through the reprogramming of keratins, in transformed cells. To address this, vimentin was stably downregulated in oral cancer derived cells. Further, global keratin profiling was performed after high salt keratin extraction. K5/K14 pair was found to be significantly downregulated, both at protein and mRNA levels upon vimentin downregulation. The previous study from our laboratory has shown a role of the K5/K14 pair in proliferation and differentiation of squamous epithelial cells. Vimentin depleted cells showed an increase in the differentiation state, marked by an increase in the levels of differentiation specific markers K1, involucrin, filaggrin and loricrin while its proliferation status remained unchanged. Rescue experiments with the K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased differentiation state. ΔNp63 emerged as one of the indirect targets of vimentin, through which it modulates the expression levels of K5/K14. Further, immunohistochemistry showed a significant correlation between high vimentin-K14 expression and recurrence/poor survival in oral cancer patients. Thus, in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression. Moreover, vimentin-K14 together may prove to be the novel markers for the prognostication of human oral cancer.</p></div

Schematic representation depicting the role of vimentin in modulation of K5/K14 expression, to regulate differentiation in carcinoma-derived cells.

<p>The model shows the regulation of K5/14 expression by vimentin, perhaps through ΔNp63. Determination of vimentin-K14 status in oral tumor tissues may have clinical implications.</p

High vimentin-K14 expression correlates with poor survival in oral cancer patients.

<p>(A) (a-c) The upper panel show images (Bar: 200μm) of IHC staining for vimentin and K14 expression in cut margin tissues while lower panel (d-f) show images (Bar: 200μm) of IHC staining for vimentin and K14 expression in tumor tissues. The negative control images represent tissue sections incubated with serum from non-immunized mice in place of primary antibodies. (B) The graphical representation shows recurrence with respect to high vimentin-K14 expression and recurrence with respect to the other combinations of vimentin-K14 expression. Kaplan-Meier survival analysis (n = 91) of (C) High vs. low vimentin expression (D) High vs. low K14 expression and (E) High vimentin-K14 vs. the other combinations of vimentin-K14 expression.</p

Downregulation of vimentin resulted in a change in the global keratin profile of the oral SCC derived cell line AW13516.

<p>(A) Schematic representation of the hypothesis. As a tumor cell acquires EMT (marked by upregulation of vimentin), it undergoes transition from a more epithelial-like to a more mesenchymal-like dedifferentiated state. To achieve this transition, there could be a vimentin mediated reprogramming of the keratins which distinguish these states. (B and C) Immunofluorescence (Bar: 10 μm) and western blot analysis of vimentin knockdown (shvim1 and shvim2) and its vector control clone (pTU6vc) using an antibody against vimentin. β-actin was used as the loading control in the western blotting experiment. (D) K8 levels of vimentin knockdown and its vector control clones were analyzed using western blotting. β-actin was used as a loading control. (E) The expression of K8 does not change upon vimentin downregulation. Thus, K8 was used as the loading control for high salt keratin enriched fraction. (F) Representative images of the 2D-gel, which show changes in keratin expression in the high salt keratin enriched fractions of vimentin knockdown and its vector control clones. The black circles indicate similarly expressed while the red circles indicate differentially expressed proteins. All the experiments were repeated independently in triplicates. For all the western blot experiments, the numbers below each blot represent the relative intensity of the bands determined using densitometry.</p