Papilla Formation Research Articles

Impact of compatible and incompatible barley—Blumeria graminis f.sp. hordei interactions on chlorophyll fluorescence parameters

Interactions between different barley genotypes and the fungal pathogen Blumeria graminis f.sp. hordei (Bgh) have a specific impact on the crop physiology. Within the context of plant resistance phenotyping, it is relevant to investigate early host–pathogen interactions to avoid the crop infestation. Analyzing different parameters of the photosynthesis apparatus gives in-depth information of the plant’s health status and can be used for a spatial and temporal assessment of interaction types during plant–pathogen infestation. In the present study, experiments were performed with a near-isogenic line of barley cv. Ingrid WT (susceptible), mlo3 (papilla-based resistance) as well as a near-isogenic line of cv. Pallas, containing the Mla1 (hypersensitive response-based resistance) gene. After inoculation with Bgh isolate K1, the leaves were measured daily using chlorophyll fluorescence imaging. Inoculated, susceptible wild-type leaves showed a reduced effective quantum yield of the photosystem II (ΦPSII) already 1 day after inoculation. In accordance with the quantum yield reduction, the non-photochemical quenching (NPQ) increased, indicating thermal dissipation of excess energy. The changes of ΦPSII and NPQ represent modifications of the leaf metabolism to aid the fungal nutrition uptake, which is influenced by Bgh. By analyzing these parameters, it was also possible to indicate resistance reactions of mlo3 and Mla1 barley genotypes against Bgh. During papilla formation in mlo3 leaves, ΦPSII revealed the lowest values. In contrast, inoculated Mla1 leaves showed the lowest NPQ. The present study proofs that chlorophyll fluorescence imaging is a valuable tool for investigating early plant–pathogen interaction noninvasively. Furthermore, this phytopathology study uses chlorophyll fluorescence imaging, chlorophyll extraction and microscopic observations to characterize the interaction response of different genotypes to an Bgh infection.

Plant exosomes: using an unconventional exit to prevent pathogen entry?

The ability to ward off filamentous pathogens, such as powdery mildew fungi, is one of the best studied examples of membrane trafficking-dependent disease resistance in plants. Here, papilla formation at the site of attack is essential for the pre-invasive immunity, whereas the encasement can hamper disease post-invasively. Exosomes containing antifungal peptides and small RNAs are thought to play a vital role in forming papillae and encasements that block fungal growth. While exosomes are well described in mammals, and have been shown to play important roles in cell-cell communication regulating development and disease, their function is not well-known in plants. In this review, we focus on some of the recent discoveries on plant exosomes and try to link this information with our current understanding of how plants use this form of unconventional secretion to acquire this durable and effective form of resistance.

Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

Spectral Patterns Reveal Early Resistance Reactions of Barley Against Blumeria graminis f. sp. hordei.

Differences in early plant-pathogen interactions are mainly characterized by using destructive methods. Optical sensors are advanced techniques for phenotyping host-pathogen interactions on different scales and for detecting subtle plant resistance responses against pathogens. A microscope with a hyperspectral camera was used to study interactions between Blumeria graminis f. sp. hordei and barley (Hordeum vulgare) genotypes with high susceptibility or resistance due to hypersensitive response (HR) and papilla formation. Qualitative and quantitative assessment of pathogen development was used to explain changes in hyperspectral signatures. Within 48 h after inoculation, genotype-specific changes in the green and red range (500 to 690 nm) and a blue shift of the red-edge inflection point were observed. Manual analysis indicated resistance-specific reflectance patterns from 1 to 3 days after inoculation. These changes could be linked to host plant modifications depending on individual host-pathogen interactions. Retrospective analysis of hyperspectral images revealed spectral characteristics of HR against B. graminis f. sp. hordei. For early HR detection, an advanced data mining approach localized HR spots before they became visible on the RGB images derived from hyperspectral imaging. The link among processes during pathogenesis and host resistance to changes in hyperspectral signatures provide evidence that sensor-based phenotyping is suitable to advance time-consuming and cost-expensive visual rating of plant disease resistances.

VPS9a Activates the Rab5 GTPase ARA7 to Confer Distinct Pre- and Postinvasive Plant Innate Immunity.

Plant innate immunity can effectively prevent the proliferation of filamentous pathogens. Papilla formation at the site of attack is essential for preinvasive immunity; in postinvasive immunity, the encasement of pathogen structures inside host cells can hamper disease. Whereas papillae are highly dependent on transcytosis of premade material, little is known about encasement formation. Here, we show that endosome-associated VPS9a, the conserved guanine-nucleotide exchange factor activating Rab5 GTPases, is required for both pre- and postinvasive immunity against a nonadapted powdery mildew fungus (Blumeria graminis f. sp hordei) in Arabidopsis thaliana Surprisingly, VPS9a acts in addition to two previously well-described innate immunity components and thus represents an additional step in the regulation of how plants resist pathogens. We found VPS9a to be important for delivering membrane material to the encasement and VPS9a also plays a predominant role in postinvasive immunity. GTP-bound Rab5 GTPases accumulate in the encasement, but not the papillae, suggesting that two independent pathways form these defense structures. VPS9a also mediates defense to an adapted powdery mildew fungus, thus regulating a durable type of defense that works in both host and nonhost resistance. We propose that VPS9a plays a conserved role in organizing cellular endomembrane trafficking, required for delivery of defense components in response to powdery mildew fungi.

Ultrastructural study on the interaction between Physcomitrella patens and Botrytis cinerea

Little is known about the interactions of bryophytes with their pathogens compared with those of flowering plants. This study used the moss Physcomitrella patens and fungal pathogen Botrytis cinerea to investigate their interaction at the ultrastructural level. Infection behaviour of B. cinerea in moss tissues was observed for 1–7 days after inoculation. Some appressoria directly penetrated P. patens cytoplasm through leaf cell walls, and others entered the intercellular spaces of leaves and infected cytoplasm. Infection hyphae were observed in sclerenchyma cells of P. patens stems for the first time. The results demonstrated that the ratio of hyphae‐inoculated leaf cells to leaf numbers could be used to quantify the disease development process at 24 h after inoculation. The ultrastructural studies revealed two responses of P. patens to B. cinerea inoculation: reinforcement of cell walls, including papillae formation; and cell death. This was the first observation of papillae for P. patens–B. cinerea interaction at the ultrastructural level. Papillae in P. patens had a similar ultrastructure to those of higher plants, and may be involved in the defence response to B. cinerea infection. Thus papillae formation is probably an evolutionarily conserved defence mechanism from the early land colonization by plants. Cell death during the P. patens–B. cinerea interaction had some features of programmed cell death, with hydrogen peroxide produced in cytoplasm membranes, suggesting something like a hypersensitive response. These ultrastructural studies suggest that P. patens could be a useful system for studying phytopathogens and could provide theoretical bases for defence mechanisms in evolutionary development.

Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development

Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development

Trichomes: interaction sites of tomato leaves with biotrophic powdery mildew pathogens

The present study aimed to explore the possibility of using the type I trichomes of tomato (Solanum lycopersicum) to monitor the infection processes of powdery mildews by microscopy. Individual trichome cells of two tomato genotypes were inoculated with pathogenic and non-pathogenic powdery mildew species, Pseudoidium neolycopersici, Erysiphe trifoliorum and Podosphaera xanthii. On the trichome cells of the tomato cultivar Moneymaker, hyphae of the pathogenic Pseudoidium neolycopersici (isolates KTP-03 and KTP-04) grew vigorously; whereas hyphal growth of the non-pathogenic Erysiphe trifoliorum and Podosphaera xanthii ceased after appressorium formation, which was associated with papilla formation and hypersensitive cell death, respectively. Similar infection processes of the tested powdery mildews were seen in Moneymaker epidermal cells. Therefore, tomato trichomes are suitable for analysing, at individual cell level, the infection processes of different pathotypes of powdery mildews and for observing the cytological responses of plants by non-pathogenic powdery mildews. On the other hand, it was observed that both isolates KTP-03 and KTP-04 failed to produce conidiophores on the hyphae elongating on Moneymaker trichomes. Similarly, no conidiophores were produced on the hyphae elongating on trichomes of Solanum peruvianum LA2172, which is resistant to KTP-03 and susceptible to KTP-04. Interestingly, delayed cell death occurred in LA2172 epidermal cells, which were attacked by KTP-03 hyphae elongating from trichomes and conidiophores were formed on new hyphae growing from the leaf epidermal cells. Thus, leaf trichomes and epidermal cells of the wild tomato species LA2172 reacted differently to the avirulent isolate KTP-03.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Scanning Electron Microscopic Study of the Tongue Papillae Formation

Objectives: To clarify the morphology of the tongue papillae formation and the metabolic characteristics along the papillae distribution in rats. Methods: The experiment was designed as a cross-sectional study and included; a scanning electron and light microscopic examination, connective tissue core analysis, and an enzyme activity test on rat tongue samples of the embryonic (n = 60) and postnatal (n = 12) periods. Results: The primordium of fungiform papillae (FuP) was already formed on embryonic day 15 (E15), and filiform papillae (FiP) was formed on E17. In the postnatal period, the middle and posterior parts of the tongue quickly developed toward the anterior part of the oral cavity. Three types of FiP were found: round, conical and branched. According to the enzyme activity test, all energy, lipid, protein and sugar metabolic enzymes showed significant differences along the tongue parts. Conclusion: No positional difference was found in the morphology of rat tongue papillae during the embryonic period. During the postnatal period, the morphology and enzyme activities of the tongue mucosa displayed differences expressing tongue development and its functional characteristics among the described tongue parts—listed above.

A New Chytridiomycete Fungus Intermixed with Crustacean Resting Eggs in a 407-Million-Year-Old Continental Freshwater Environment.

The 407-million-year-old Rhynie Chert (Scotland) contains the most intact fossilised remains of an early land-based ecosystem including plants, arthropods, fungi and other microorganisms. Although most studies have focused on the terrestrial component, fossilised freshwater environments provide critical insights into fungal-algal interactions and the earliest continental branchiopod crustaceans. Here we report interactions between an enigmatic organism and an exquisitely preserved fungus. The fungal reproductive structures are intermixed with exceptionally well-preserved globular spiny structures interpreted as branchiopod resting eggs. Confocal laser scanning microscopy enabled us to reconstruct the fungus and its possible mode of nutrition, the affinity of the resting eggs, and their spatial associations. The new fungus (Cultoraquaticus trewini gen. et sp. nov) is attributed to Chytridiomycota based on its size, consistent formation of papillae, and the presence of an internal rhizoidal system. It is the most pristine fossil Chytridiomycota known, especially in terms of rhizoidal development and closely resembles living species in the Rhizophydiales. The spiny resting eggs are attributed to the crustacean Lepidocaris rhyniensis, dating branchiopod adaptation to life in ephemeral pools to the Early Devonian. The new fungal interaction suggests that, as in modern freshwater environments, chytrids were important to the mobilisation of nutrients in early aquatic foodwebs.

English

Many quantitative trait loci (QTLs) in different barley populations were discovered for resistance to Puccinia hordei and heterologous rust species. Partial resistance (host basal resistance) and nonhost resistance (nonhost basal resistance) in barley to leaf rusts are based on prehaustorial mechanism of resistance which is associated with papillae formation. They are mainly governed by genes with relatively small, quantitative effects, located on QTL. The genes for host basal resistance seem to play similar roles in basal resistance as those governing nonhost basal resistances. From different studies it was observed that these two resistance types are based on shared principles. Four and two quantitative trait loci-near isogenic lines (QTL-NILs), respectively, were developed for basal resistance and nonhost resistance QTLs using SusPtrit as recurrent parent. SusPtrit is a research line which is exceptionally susceptible to leaf rusts for which normally barley is a nonhost. They were infected with one homologous ( P. hordei isolate 1.2.1) and three heterologous ( Puccinia triticina isolate ‘Flamingo’, P. hordei -murini and P. hordei -secalini) leaf rusts in three replications at seedling stage to evaluate whether relatively large-effect QTLs show specificity in their reaction to homologous and heterologous rust isolates. The result showed that, the QTLs for host basal resistance and the QTL for nonhost basal resistance have a significant (P<0.05) effect on both homologous and heterologous rusts. Also, higher positive (P<0.05, r = 0.98) correlation was observed between different macroscopic and microscopic parameters measured indicating that there is a possible association between partial resistance quantitative trait loci and nonhost resistance quantitative trait loci. These give an indication that indeed, host and nonhost basal resistance are associated. Keywords: Partial resistance, nonhost resistance, near isogenic lines, barley

Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development

Grainyhead-like 3 (Grhl3) is a transcription factor involved in epithelial morphogenesis. In the present study, we evaluated the developmental role of Grhl3 in structural formation of the circumvallate papilla (CVP), which undergoes dynamic morphological changes during organogenesis. The specific expression pattern of Grhl3 was examined in the CVP-forming region, specifically in the apex and epithelial stalk from E13.5 to E15.5 using in situ hybridization. To determine the role of Grhl3 in epithelial morphogenesis of the CVP, we employed an in vitro tongue culture method, wherein E13.5 tongue were isolated and cultured for 2days after knocking down of Grhl3. Knockdown of Grhl3 resulted in significant changes to the epithelial structure of the CVP, such that the apical region of the CVP was smaller in size, and the epithelial stalks were more deeply invaginated. To define the mechanisms underlying these morphological alterations, we examined cell migration, proliferation, and apoptosis using phalloidin staining, immunohistochemistry against Ki67, ROCK1, and E-cadherin, and a TUNEL assay, respectively. These results revealed an increase in proliferation, a reduction in apoptosis, and an altered pattern of cytoskeletal formation in the CVP-forming epithelium, following Grhl3 knockdown. In addition, there were changes in the specific expression patterns of signaling and apoptosis-related molecules such as Axin2, Bak1, Bcl2, Casp3, Casp8, Ctnnb1, Cnnd1, Gli3, Lef1, Ptch1, Rock1, Shh, and Wnt11, which could explain the altered cellular and morphological events. Based on these results, we propose that developmental stage-specific Grhl3 plays a significant role in CVP morphogenesis not by just disruption of epithelial integrity but by regulating epithelial cell proliferation, apoptosis, and migration via Shh, Wnt, and apoptosis signaling during mouse embryogenesis.

Attenuation of PAMP-triggered immunity in maize requires down-regulation of the key β-1,6-glucan synthesis genes KRE5 and KRE6 in biotrophic hyphae of Colletotrichum graminicola.

In plants, pathogen defense is initiated by recognition of pathogen-associated molecular patterns (PAMPs) via plasma membrane-localized pattern-recognition receptors (PRRs). Fungal structural cell wall polymers such as branched β-glucans are essential for infection structure rigidity and pathogenicity, but at the same time represent PAMPs. Kre5 and Kre6 are key enzymes in β-1,6-glucan synthesis and formation of branch points of the β-glucan network. In spite of the importance of branched β-glucan for hyphal rigidity and plant-fungus interactions, neither the role of KRE5 and KRE6 in pathogenesis nor mechanisms allowing circumventing branched β-glucan-triggered immune responses are known. We functionally characterized KRE5 and KRE6 of the ascomycete Colletotrichum graminicola, a hemibiotroph that infects maize (Zea mays). After appressorial plant invasion, this fungus sequentially differentiates biotrophic and highly destructive necrotrophic hyphae. RNAi-mediated reduction of KRE5 and KRE6 transcript abundance caused appressoria to burst and swelling of necrotrophic hyphae, indicating that β-1,6-glucosidic bonds are essential in these cells. Live cell imaging employing KRE5:mCherry and KRE6:mCherry knock-in strains and probing of infection structures with a YFP-conjugated β-1,6-glucan-binding protein showed expression of these genes and exposure of β-1,6-glucan in conidia, appressoria and necrotrophic, but not in biotrophic hyphae. Overexpression of KRE5 and KRE6 in biotrophic hyphae led to activation of broad-spectrum plant defense responses, including papilla and H2 O2 formation, as well as transcriptional activation of several defense-related genes. Collectively, our results strongly suggest that down-regulation of synthesis and avoidance of exposure of branched β-1,3-β-1,6-glucan in biotrophic hyphae is required for attenuation of plant immune responses.

Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and resistance mechanism.

Stress conditions in agricultural ecosystems can occur at variable intensities. Different resistance mechanisms against abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100, and 150mM NaCl) on powdery mildew resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as near-isogenic lines (NILs) carrying the resistance gene Ol-1 (associated with a slow hypersensitivity response; HR), ol-2 (an mlo mutant associated with papilla formation), and Ol-4 (an R gene associated with a fast HR). Powdery mildew resistance was affected by salt stress in a genotype- and stress intensity-dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied by accelerated cell death-like senescence. In contrast, severe salt stress (150mM) reduced disease symptoms. Na(+) and Cl(-) accumulation in the leaves was linearly related to the decreased pathogen symptoms under severe stress. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress in NIL-Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes and the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant's performance under the combination of abiotic and biotic stress.

Correspondence between symptom development of Colletotrichum graminicola and fungal biomass, quantified by a newly developed qPCR assay, depends on the maize variety.

BackgroundPenetration attempts of the hemibiotroph Colletotrichum graminicola may activate PAMP-triggered immunity (PTI) on different cultivars of Zea mays to different extent. However, in most events, this does not prevent the establishment of a compatible pathogenic interaction. In this study, we investigate the extent to which the host variety influences PTI. Furthermore, we assess whether visual disease symptoms occurring on different maize varieties reliably reflect fungal biomass development in planta as determined by qPCR and GFP tracing.ResultsEmploying a set of four maize varieties, which were selected from a panel of 27 varieties, for in-depth assessment of pathogenesis of the wild type strain of C. graminicola, revealed considerable differences in susceptibility as evidenced by symptom severity that decreased from variety Golden Jubilee to Mikado to Farmtop to B73. However, a newly developed qPCR assay and microscopical observation of a GFP-labelled strain showed that disease symptoms are in some instances inconsistent when compared with other indicators of susceptibility. Of the four varieties assessed, either Golden Jubilee, Mikado and B73, or Golden Jubilee, Farmtop and B73 showed a direct correlation between symptom and fungal biomass development. In a pairwise comparison, however, Mikado and Farmtop showed an inverse correlation for these features.ConclusionsThe genotype of maize contributes to the severity of symptoms resulting from an infection with C. graminicola. Partially, this may be attributed to the extent of PTI activated in different varieties, as reflected by papilla formation. Furthermore, when evaluating the susceptibility of a variety, it should be considered that symptom severity must not have to reflect the extent of fungal growth in the infected tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0709-4) contains supplementary material, which is available to authorized users.

The usefulness of measuring tear periostin for the diagnosis and management of ocular allergic diseases

Chronic ocular allergic diseases such as vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) are accompanied by serious comorbidities; however, the underlying pathogenesis remains obscure. Furthermore, diagnosing conjunctival lesions in patients with atopic dermatitis and estimating the severity in AKC are important for the treatment of ocular allergic diseases. We addressed whether periostin, a novel mediator and biomarker in allergic inflammation, is involved in the pathogenesis of ocular allergic diseases and whether periostin can be a biomarker for these diseases. We investigated tear periostin in patients with seasonal allergic conjunctivitis (SAC), VKC, and AKC and allergic patients without conjunctivitis and compared it with tear IL-13 and serum periostin. Furthermore, in patients with AKC, we measured tear periostin before and after topical treatment with tacrolimus. Tears from patients with ocular allergic disease showed significantly high periostin levels than did tears from allergic patients without conjunctivitis and from patients with AKC, VKC, and SAC in descending order. Tear periostin was associated with serious comorbidities such as large papilla formation and corneal damage in AKC, although both tear IL-13 and serum periostin had little to no such abilities. Furthermore, after topical tacrolimus treatment, tear periostin tended to decrease in most patients with AKC along with their clinical improvement. Periostin produced in conjunctival tissues stimulated by IL-13 may contribute to the pathogenesis of ocular allergic diseases. Furthermore, tear periostin can be potentially applied as a biomarker to diagnose conjunctivitis in allergic patients and to evaluate disease severity as well as the efficacy of treatments in AKC.

The use of pulsed CO2 lasers for the treatment of vulvovaginal atrophy.

This article reviews the literature regarding the safety and efficacy of the use of a pulsed CO2 laser for the treatment of vulvovaginal atrophy (VVA). Prospective observational studies have demonstrated histological changes after the use of pulsed CO2 laser vaginally in atrophic conditions. Increased collagen and extracellular matrix production has been reported together with an increase in the thickness of the vaginal epithelium with the formation of new papilla. Three different observational studies reported a significant improvement of VVA assessed subjectively (with a 10-point visual analogue scale) and objectively (using the Vaginal Health Index) after a cycle of three treatments of pulsed CO2 laser. Also sexual function (assessed with the Female Sexual Function Index) and quality of life (evaluated with the SF12 questionnaire) significantly improved. No complications or side-effects were reported during or after the laser procedure that was performed in an outpatient setting. Increasing evidence with histological and clinical data supports the use of pulsed CO2 lasers in the treatment of VVA; however, no randomized control trial (sham versus treatment) has yet been produced and no data on the duration of therapy are currently available.

Immediate loading of dental implants with simultaneous formation of gingival papillae

Immediate loading of dental implants with simultaneous formation of gingival papillae

MicroRNA let-7b-regulated epidermal stem cell proliferation in hypertrophied anal papillae.

The present study investigated the role of epidermal stem cell-expressed microRNA let-7b in the pathogenesis of hypertrophied anal papillae. Hypertrophied anal papillae were examined for the presence of epidermal stem cells. Epidermal stem cells were identified using flow cytometry and immunofluorescent staining for the cell surface markers, integrin α6 and integrin β1 subunits. Expression levels of microRNA let-7b in α6+/β1+and α6-/β1-cells were compared using reverse transcription-quantitative polymerase chain reaction and northern blotting. Lentivirus-mediated expression of microRNA let-7b in epidermal stem cells was utilized in order to study the effects of this microRNA on the cell cycle proteins, cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4). MicroRNA let-7b-overexpressing cells were examined using flow cytometry, in order to determine the effects of the microRNA on cell cycle progression. α6+/β1+epidermal stem cells were identified in hypertrophic anal papillae. Following isolation and enrichment of the α6+/β1+population, these cells were found to have a rapid rate of proliferation in vitro. The expression of cell cycle-related proteins was elevated in this population, compared with that in α6-/β1-cells. The expression of microRNA let-7b in α6+/β1+epidermal stem cells was significantly lower than that in α6-/β1-cells. Two microRNA let-7b target genes, CCND1 and CDK4, were found to be upregulated in α6+/β1+cells. When the exogenous precursor, microRNA let-7, was overexpressed in α6+/β1+ epidermal stem cells, the cell proliferation rate was significantly lower than that in cells expressing microRNA let-7 containing a mutated seed sequence. The addition of exogenous microRNA let-7 resulted in an increased expression level of mature microRNA let-7b, while the expression of CCND1 and CDK4 was reduced. Epidermal stem cells transfected with microRNA let-7b were arrested in the G2/M phase and the percentage of cells in S-phase was significantly reduced. In conclusion, let-7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.