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VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p &lt, 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.

The Merkel cell-neurite complex initiates the perception of touch and mediates Aβ slowly adapting type I responses. Lichen planus is a chronic inflammatory autoimmune disease with T-cell-mediated inflammation, whereas hyperkeratosis is characterized with or without epithelial dysplasia in the oral mucosa. To determine the effects of lichen planus and hyperkeratosis on the Merkel cell-neurite complex, healthy oral mucosal epithelium and lesional oral mucosal epithelium of lichen planus and hyperkeratosis patients were stained by immunohistochemistry (the avidin-biotin-peroxidase complex and double immunofluorescence methods) using pan cytokeratin, cytokeratin 20 (K20, a Merkel cell marker), and neurofilament 200 (NF200, a myelinated Aβ- and Aδ-nerve fibre marker) antibodies. NF200-immunoreactive (ir) nerve fibres in healthy tissues and in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis were counted and statistically analysed. In the healthy oral mucosa, K20-positive Merkel cells with and without close association to the intraepithelial NF200-ir nerve fibres were detected. In the lesional oral mucosa of lichen planus and hyperkeratosis patients, extremely rare NF200-ir nerve fibres were detected only in the lamina propria. Compared with healthy tissues, lichen planus and hyperkeratosis tissues had significantly decreased numbers of NF200-ir nerve fibres in the oral mucosal epithelium. Lichen planus and hyperkeratosis were associated with the absence of Aβ-nerve endings in the oral mucosal epithelium. Thus, we conclude that mechanosensation mediated by the Merkel cell-neurite complex in the oral mucosal epithelium is impaired in lichen planus and hyperkeratosis.

Histone deacetylases (HDACs) are components of nuclear multiprotein complexes that deacetylate histones and perform important roles in repression of transcription. Using specific rabbit mAbs, we analyzed by immunohistochemistry and confocal immunofluorescence analysis the expression and subcellular localization of HDAC1–4 and HDAC9 in sections of adult human third molars. HDAC2 and HDAC9 were expressed in some pulpal cells and strongly expressed in the majority of mature odontoblasts. In contrast, only weak expression of HDAC1, HDAC3 and HDAC4 was observed. Confocal immunofluorescence analysis together with the DNA stain DRAQ5 revealed that HDAC2 and HDAC9 were coexpressed within the odontoblast nucleus, but localized to distinct subnuclear structures. In contrast to the current point of view, HDAC2 is strongly expressed in a terminally differentiated cell type. Our results imply that class I and II HDACs are involved in the transcriptional regulation of human odontoblasts in vivo.

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α1β1, α2β1) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α1-, β1-, and α2-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α1-, β1-, and α2-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α1-, β1-, and α2-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization.Decalcified free-floating sections of rat molars with PDL were incubated using total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p-ERK1/2. To test whether p-ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p-ERK1/2 with calcitonin gene-related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point-counting method.In cervical, midroot, and apical zones of the PDL, t-ERK1/2- and p-ERK1/2-labeled nerve fibers were found in close association with blood vessels. The p-ERK1/2-labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p-ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin.The perivascular distribution of t-ERK1/2 and p-ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p-ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.

The epithelial rests of Malassez (ERM) are derived from the Hertwig's epithelial root sheath during tooth development. The ERM contain endothelial nitric oxide synthase (eNOS), but the existence of phosphorylation site/s of eNOS in the ERM is unclear.Rat molars with periodontium were perfusion- and post-fixed, decalcified, and frozen-sectioned. Free-floating sections were incubated using antisera against total eNOS, eNOS phosphorylated at serine (Ser)1177, Ser116, and threonine (Thr)495. The signal intensities of t-eNOS, p-eNOS at Ser1177 and Ser116 in the ERM were measured by densitometry and statistically analyzed.In the ERM, localization of total eNOS and the phosphorylation sites of eNOS at Ser1177 and Ser116 were detected, while a basal localization of eNOS phosphorylated at Thr495 in the ERM was undetectable. For p-eNOS at Ser116 regional differences in phosphorylation were detected.The basal production of NO by eNOS in the ERM is modulated by phosphorylation of eNOS at Ser1177 and Ser116 residues, while the basal activity of the eNOS is not influenced by phosphorylation of eNOS at Thr495 residue. This provides evidence that phosphorylation plays a key role for regulation of the catalytic activity of eNOS.

Nitric oxide (NO) mediates different cellular functions by activating soluble guanylate cyclase (sGC) that converts guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophosphate (cGMP). Membrane-bound GCs produce cGMP in response to natriuretic peptides in osteoblasts, but neither the NO-target enzyme sGC, nor the phosphorylation sites of NOS III, nor their regulation by extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt/protein kinase B (Akt/PKB) in osteoclasts have been established.Rat molars with periodontium were perfusion- and post-fixed, decalcified, and frozen-sectioned. Free-floating sections were stained using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and tartrate-resistant acid phosphatase (TRAP) histochemical techniques and immunoreacted with antisera against NO-synthase (NOS) I-III, NOS III phoshorylated at Thr495, NOS III phoshorylated at Serine1177 (Ser1177), ERK1/2, phosphorylated ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2/beta1), and cGMP.NADPH-d staining and immunostaining of NOS I-III, NOS III phosphorylated at Ser1177, ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2 and beta1-subunits), and cGMP were detected in osteoclasts. Immunohistochemical reaction products for NOS III phosphorylated at threonine495 (Thr495) and phosphorylated ERK1/2 could not be identified in osteoclasts. Comparison of TRAP activity and immunostaining for sGC beta1-subunit revealed that sGC beta1-subunit is only expressed in a sub-population of osteoclasts.NO is likely to be generated by NOS I and NOS III in osteoclasts. The inconstant expression of NOS II in some osteoclasts may be explained with inducible expression of NOS II upon physiological cell activation. Localization of the sGC alpha2- and beta1-subunits and cGMP in osteoclasts is compatible with an involvement of NO-sGC signaling in the homeostasis of osteoclasts. The phosphorylation site of NOS III at Ser1177 and phosphorylated Akt/PKB are involved in regulation of NO production by NOS III in osteoclasts under basal conditions.

It is known that total endothelial nitric oxide synthase (eNOS) is localized in peripheral nerve fibers, but the existence of the phosphorylation site/s of eNOS in peripheral nerve fibers is unknown. In the perfusion-fixed and decalcified sections of rat palates, eNOS and eNOS phosphorylated at Ser(116) were identified in the nerve fibers of the glandulae palatinae. The localization of eNOS phosphorylated at Ser(1177) and Thr(495) in nerve fibers was undetectable. It is concluded that eNOS is phosphorylated at Ser(116) in nerve fibers of the glandulae palatinae under basal conditions. The basal Ser(1177) and Thr(495) phosphorylation of eNOS are missing in nerve fibers of the glandulae palatinae.

A wide variety of stimuli can trigger activation of the transcription factor CREB (cAMP-responsive element binding protein), pointing toward a central role for CREB in the integration of various signaling inputs. No data are available on the expression and phosphorylation of CREB in mammalian teeth. Using immunohistochemical analysis of free-floating sections, we show here that CREB was strongly expressed and phosphorylated at Ser-133 within the nucleus of a subpopulation of adult human molar odontoblasts. Many dental pulp stromal cells and periodontal ligament fibroblasts expressed CREB and showed phosphorylation of CREB at Ser-133. In addition, cementoblasts displayed nuclear expression and phosphorylation of CREB at Ser-133. The epithelial rests of Malassez revealed strong nuclear expression of CREB, but phosphorylation at Ser-133 was variable. Our results provide the first evidence that the constitutively phosphorylated transcription factor CREB is involved in the biomineralization process of adult human molar odontoblasts and cementoblasts.

BackgroundWhile ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³) results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise.PurposeDetermine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers.Methods7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques.ResultsWhile total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (pConclusionResistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and concomitantly pAMPKThr¹⁷² levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser²⁸⁴³ can mechanistically impact Ca²⁺handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca(2+)-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca(2+)-binding proteins in the ERM has not so far been characterized. Among the three Ca(2+)-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca(2+) is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca(2+) is regulated only by calretinin. The expression of Ca(2+)-binding proteins is restricted in a developmental manner in the ERM.

The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBαat Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBαwas detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBαis dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBαas well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit.

Although prion protein’s (PrP) involvement in transmission of degenerative neurological diseases has been subjected to considerable scrutiny, its physiological role is still obscure. The distribution of PrP in dental tissues was investigated using three different methods: immunohistochemistry, cell culture, and scanning electron microscopy. PrP knockout mice were found to have marked anomalies in dentin structure. In human teeth, cementoblasts and odontoblasts showed prominent staining for PrP at levels comparable to those of nerve fibers. Epithelial rests of Malassez, which are remnants of a cell type formerly forming enamel, were also positive. Thus, all PrP-positive cells in human dentition are in some way involved in calcified tissue formation. This suggests a previously undetected function of prion protein in healthy vertebrates as evidenced by an obvious phenotype in PrP knockout mice. Periodontal and pulpal tissue exposed by disease or trauma might represent a clinically relevant entry point for prions incorporated orally and thus a possible mode of infection.

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10−7 M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10−6 M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

Large clinical studies provided evidence of a lower incidence of a reduced cardiac morbidity in diabetic patients treated with angiotensin type 1 (AT1) receptor blockers. The AT1 receptor is coupled to the Gq class of G-proteins, which stimulate proteinkinase C (PKC) via activation of phospholipase Cβ. To study the role of the Gq protein Gα11 and its signaling through PKC in diabetic heart disease, we induced diabetes in wildtype and Gα11 knockout mice using streptozotocin.After eight weeks of stable hyperglycemia, cardiac morphology and function were assessed by echocardiography, myocardial expression and translocation of PKC isoforms by immunohistochemistry and immunoblot after tissue fractionation.Wildtype mice (n=8) but not Gα11 knockout animals (n=8) developed ventricular hypertrophy upon induction of hyperglycemia. PKC isoforms α, βII, δ, e, ζ and θ were all detected in myocardium of wildtype animals. Compared to normoglycemic control animals, PKC isoforms α and ζ showed increased expression levels in diabetic wildtype mice. In addition, PKC ζ was phosphorylated at Thr410/403 and showed strong translocation to nuclear membranes in cardiomyocytes of diabetic but not of control animals. Hearts from normoglycemic Gα11-knockout mice showed lower expression levels of PKC α and δ compared to wildtype animals. Upon induction of hyperglycemia in knockouts, expression of PKC δ was only slightly increased to the level found in non-diabetic wildtypes. Expression, phosphorylation and translocation of PKC isoforms, however, remained unaffected by the induction of diabetes in Gα11-deficient mice.We conclude that Gα11 is central to the induction of myocardial hypertrophy in type 1 diabetes. Activation of PKC α and ζ appear to be important pathways in hypertrophic signaling via Gα11. The inhibition of this pathway may in part explain the strong therapeutic benefit of AT1 receptor blockade in diabetic patients.

By the formation of cyclic guanosine 3′,5′-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC α2-subunit/β1-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC α2, and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10−5 M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10−4 M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.