Spatio‐Temporal Parameters of Endosomal Signaling in Cancer: Implications for New Treatment Options

The endo/lysosomal system in cells provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction, thus, is essential for physiological signaling. Endocytic organelles can significantly extend signaling of activated cell surface receptors, and may additionally provide distinct locations for the generation of specific signaling outputs. Failures of regulation at different levels of endocytosis, recycling, degradation as well as aberrations in specific endo/lysosomal signaling pathways, such as mTORC1, might lead to different diseases including cancer. Therefore, a better understanding of spatio‐temporal compartmentalization of sub‐cellular signaling might provide an opportunity to interfere with aberrant signal transduction in pathological processes by novel combinatorial therapeutic approaches. J. Cell. Biochem. 117: 836–843, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

DMSO‐ and Serum‐Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord

The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post‐thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [?1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post‐thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog‐Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin‐V‐positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv‐Cock). Real‐time PCR and Western blot analysis of post‐thaw WJMSCs from Conv‐Cock group showed significantly increased expression of pro‐apoptotic factors (BAX, p53, and p21) and reduced expression of anti‐apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog‐Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC‐based regenerative therapies. J. Cell. Biochem. 117: 2397–2412, 2016. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

     

Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Lovastatin‐Mediated Changes in Human Tendon Cells

Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol‐lowering and anti‐atherosclerotic properties. Although some statin patients may experience muscle‐related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first‐pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin‐related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon‐specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short‐term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP‐2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose‐dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function. J. Cell. Physiol. 230: 2543–2551, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Detection and Characterization of the Effect of AB‐FUBINACA and Its Metabolites in a Rat Model

Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB‐FUBINACA is an example of one of the new indazole‐carboxamide synthetic cannabinoids introduced in the past year. Even though AB‐FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB‐FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB‐FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real‐time quantitative RT‐PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB‐FUBINACA in urine and its effect on the gene expressions in liver and heart. J. Cell. Biochem. 117: 1033–1043, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals. Inc.     

“Nuclear FGF Receptor‐1 and CREB Binding Protein: An Integrative Signaling Module”

In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor‐1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene‐controlling Feed‐Forward‐And‐Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation. J. Cell. Physiol. 230: 989–1002, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Anaplerotic Accumulation of Tricarboxylic Acid Cycle Intermediates as Well as Changes in Other Key Metabolites During Heterotopic Ossification

Heterotopic ossification (HO) is the de novo formation of bone that occurs in soft tissue, through recruitment, expansion, and differentiation of multiple cells types including transient brown adipocytes, osteoblasts, chondrocytes, mast cells, and platelets to name a few. Much evidence is accumulating that suggests changes in metabolism may be required to accomplish this bone formation. Recent work using a mouse model of heterotopic bone formation reliant on delivery of adenovirus‐transduced cells expressing low levels of BMP2 showed the immediate expansion of a unique brown adipocyte‐like cell. These cells are undergoing robust uncoupled oxidative phosphorylation to a level such that oxygen in the microenvironment is dramatically lowered creating areas of hypoxia. It is unclear how these oxygen changes ultimately affect metabolism and bone formation. To identify the processes and changes occurring over the course of bone formation, HO was established in the mice, and tissues isolated at early and late times were subjected to a global metabolomic screen. Results show that there are significant changes in both glucose levels, as well as TCA cycle intermediates. Additionally, metabolites necessary for oxidation of stored lipids were also found to be significantly elevated. The complete results of this screen are presented here, and provide a unique picture of the metabolic changes occurring during heterotopic bone formation. J. Cell. Biochem. 117: 1044–1053, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

ACTH Modulates PTP‐PEST Activity and Promotes Its Interaction With Paxillin

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in‐gel PTP assays have shown the activation of a 115‐kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP‐PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP‐PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT‐PCR studies revealed PTP‐PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP‐PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non‐ACTH‐treated rats increased K_M and V_M. Finally, in‐gel PTP assays of immunoprecipitated paxillin from control and ACTH‐treated rats suggested a hormone‐mediated increase in paxillin–PTP115 interaction, while PTP‐PEST and paxillin co‐localize in Y1 cells. Taken together, these data demonstrate PTP‐PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH‐induced PTP–PEST–paxillin interaction. J. Cell. Biochem. 117: 2170–2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Ghrelin Augments the Expressions and Secretions of Proinflammatory Adipokines,VEGF120 and MCP‐1, in Differentiated 3T3‐L1 Adipocytes

Ghrelin is a physiological‐active peptide with growth hormone‐releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin‐direct‐effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3‐L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9‐fold increase on vascular endothelial growth factor‐120 (VEGF₁₂₀) releases (p < 0.01) and the 1.7‐fold on monocyte chemoattractant protein‐1 (MCP‐1) (p < 0.01) from 3T3‐L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, IL‐10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c‐Jun NH₂‐terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4‐fold (p < 0.01) and 1.6‐fold (p < 0.01) in the ghrelin‐stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3‐kinase (PI3K), significantly decreased the amplified VEGF₁₂₀ secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP‐1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP‐1, but not VEGF₁₂₀, release by 35% relative to the only ghrelin‐stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF₁₂₀ and MCP‐1, but fails to affect IL‐10 and adiponectin which are considered to be anti‐inflammatory adipokines. Moreover, this augmented VEGF₁₂₀ release is invited through the activation of PI3K pathways and the MCP‐1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct‐action in peripheral tissues as well as via in CNS. J. Cell. Physiol. 230: 199–209, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

The Role of Auxiliary Subunits for the Functional Diversity of Voltage‐Gated Calcium Channels

Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α₁ subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc.     

Induced conformational change in human IL‐4 upon binding of a signal‐neutralizing DARPin

The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97 , 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ_c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.