Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

Granzyme serine proteases in inflammation and rheumatic diseases.

Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation.

How health literacy is taught and evaluated in dentistry, medicine, nursing, law, pharmacy, public health, and social work: a narrative review.

BACKGROUND: This narrative review examined the published peer-reviewed literature on how health literacy is taught and evaluated in seven health professional and adjacent disciplines: dentistry, medicine, nursing, law, pharmacy, public health, and social work. The study objectives were to assess how students are educated about health literacy and how their health literacy education and skills are evaluated. METHODS: Study selection followed guidelines outlined in PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). We searched PubMed, CINAHL, SocINDEX (EBSCOhost), Lexis Advance and Public Health (ProQuest) for English-language publications of health literacy education studies across seven disciplines at U.S.-based institutions. Inclusion criteria included: 1) methods describing a primary health literacy educational intervention, 2) professional education in one or more of the seven disciplines, 3) educational institutions in the United States, and 4) articles published in peer-reviewed journals between 2000 and 2020. RESULTS: The searches yielded 44 articles. Health literacy education is evident in six of the seven studied disciplines, and varies widely in the quality, quantity, timing and mode of education and evaluation. Despite the presence of health literacy accreditation requirements, none of the seven disciplines has developed and implemented a standard, rigorous health literacy education program for students. CONCLUSIONS: Graduating institutions and professional accreditation organizations that set the standards for education must lead the way by implementing upstream changes in health literacy professional education. Teaching health literacy to students in health professions is one strategy to help close gaps in patient/client professional communication for graduates and those they serve.

Granzyme B as a therapeutic target: an update in 2022.

INTRODUCTION: Granzyme B is a serine protease extensively studied for its implication in cytotoxic lymphocyte-mediated apoptosis. In recent years, the paradigm that the role of granzyme B is restricted to immune cell-mediated killing has been challenged as extracellular roles for the protease have emerged. While mostly absent from healthy tissues, granzyme B levels are elevated in several autoimmune and/or chronic inflammatory conditions. In the skin, its accumulation significantly impairs proper wound healing. AREAS COVERED: After an overview of the current knowledge on granzyme B, a description of newly identified functions will be presented, focussing on granzyme B ability to promote cell-cell and dermal-epidermal junction disruption, extracellular matrix degradation, vascular permeabilization, and epithelial barrier dysfunction. Progress in granzyme B inhibition, as well as the use of granzyme B inhibitors for the treatment of tissue damage, will be discussed. EXPERT OPINION: The absence of endogenous extracellular inhibitors renders extracellular granzyme B accumulation deleterious for the proper healing of chronic wounds due to sustained proteolytic activity. Consequently, specific granzyme B inhibitors have been developed as new therapeutic approaches. Beyond applications in wound healing, other autoimmune and/or chronic inflammatory conditions related to exacerbated granzyme B activity may also benefit from the development of these inhibitors.

Noncytotoxic Roles of Granzymes in Health and Disease.

Granzymes are serine proteases previously believed to play exclusive and somewhat redundant roles in lymphocyte-mediated target cell death. However, recent studies have challenged this paradigm. Distinct substrate profiles and functions have since emerged for each granzyme while their dysregulated proteolytic activities have been linked to diverse pathologies.

Granzyme B in epithelial barrier dysfunction and related skin diseases.

The predominant function of the skin is to serve as a barrier-to protect against external insults and to prevent water loss. Junctional and structural proteins in the stratum corneum, the outermost layer of the epidermis, are critical to the integrity of the epidermal barrier as it balances ongoing outward migration, differentiation, and desquamation of keratinocytes in the epidermis. As such, epidermal barrier function is highly susceptible to upsurges of proteolytic activity in the stratum corneum and epidermis. Granzyme B is a serine protease scarce in healthy tissues but present at high levels in tissues encumbered by chronic inflammation. Discovered in the 1980s, granzyme B is currently recognized for its intracellular roles in immune cell-mediated apoptosis as well as extracellular roles in inflammation, chronic injuries, tissue remodeling, as well as processing of cytokines, matrix proteins, and autoantigens. Increasing evidence has emerged in recent years supporting a role for granzyme B in promoting barrier dysfunction in the epidermis by direct cleavage of barrier proteins and eliciting immunoreactivity. Likewise, granzyme B contributes to impaired epithelial function of the airways, retina, gut, and vessels. In the present review, the role of granzyme B in cutaneous epithelial dysfunction is discussed in the context of specific conditions with an overview of underlying mechanisms as well as utility of current experimental and therapeutic inhibitors.

Inhibitors of PARP: Number crunching and structure gazing.

SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.

High Myc expression and transcription activity underlies intra-tumoral heterogeneity in triple-negative breast cancer.

We have previously identified a novel intra-tumoral dichotomy in triple-negative breast cancer (TNBC) based on the differential responsiveness to a reporter containing the Sox2 regulatory region-2 (SRR2), with reporter responsive (RR) cells being more stem-like than reporter unresponsive (RU) cells. Using bioinformatics, we profiled the protein-DNA binding motifs of SRR2 and identified Myc as one of the potential transcription factors driving SRR2 activity. In support of its role, Myc was found to be highly expressed in RR cells as compared to RU cells. Enforced expression of MYC in RU cells resulted in a significant increase in SRR2 activity, Myc-DNA binding, proportion of cellsexpressing CD44+/CD24-, chemoresistance and mammosphere formation. Knockdown of Myc using siRNA in RR cells led to the opposite effects. We also found evidence that the relatively high ERK activation in RR cells contributes to their high expression of Myc and stem-like features. Using confocal microscopy and patient samples, we found a co-localization between Myc and CD44 in the same cell population. Lastly, a high proportion of Myc-positive cells in tumors significantly correlated with a short patient survival. In conclusion, inhibition of the MAPK/ERK/Myc axis may be an effective approach in eliminating stem-like cells in TNBC.

Elevated ARG1 expression in primary monocytes-derived macrophages as a predictor of radiation-induced acute skin toxicities in early breast cancer patients.

Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their "M1/M2" activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0 .032), moist desquamation (p = 0 .027), and CTC grade (p = 0 .056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0 .069), moist desquamation (p = 0 .037), and CTC grade (p = 0 .046). To conclude, our findings underline for the first time the biological significance of increased ARG1 mRNA levels as an early independent predictive biomarker of RT-induced acute skin toxicities.

Triple negative breast cancers comprise a highly tumorigenic cell subpopulation detectable by its high responsiveness to a Sox2 regulatory region 2 (SRR2) reporter.

We have recently described a novel phenotypic dichotomy within estrogen receptor-positive breast cancer cells; the cell subset responsive to a Sox2 regulatory region (SRR2) reporter (RR cells) are significantly more tumorigenic than the reporter unresponsive (RU) cells. Here, we report that a similar phenomenon also exists in triple negative breast cancer (TNBC), with RR cells more tumorigenic than RU cells. First, examination of all 3 TNBC cell lines stably infected with the SRR2 reporter revealed the presence of a cell subset exhibiting reporter activity. Second, RU and RR cells purified by flow cytometry showed that RR cells expressed higher levels of CD44, generated more spheres in a limiting dilution mammosphere formation assay, and formed larger and more complex structures in Matrigel. Third, within the CD44(High)/CD24- tumor-initiating cell population derived from MDA-MB-231, RR cells were significantly more tumorigenic than RU cells in an in vivo SCID/Beige xenograft mouse model. Examination of 4 TNBC tumors from patients also revealed the presence of a RR cell subset, ranging from 1.1-3.8%. To conclude, we described a novel phenotypic heterogeneity within TNBC, and the SRR2 reporter responsiveness is a useful marker for identifying a highly tumorigenic cell subset within the CD44(High)/CD24-tumor-initiating cell population.

Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays.

INTRODUCTION: Aberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets. METHODS: We used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples. RESULTS: We found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples. CONCLUSIONS: In conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.

YB-1 regulates Sox2 to coordinately sustain stemness and tumorigenic properties in a phenotypically distinct subset of breast cancer cells.

BACKGROUND: Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells. METHODS: Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown. RESULTS: Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets. CONCLUSIONS: To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties.

ß-Catenin, a Sox2 binding partner, regulates the DNA binding and transcriptional activity of Sox2 in breast cancer cells.

Sox2, an embryonic stem cell marker, has been recently implicated in the pathogenesis of breast cancer (BC). Using liquid chromatography-mass spectrometry and co-immunoprecipitation, we identified ß-catenin as a Sox2 binding partner in MCF7 cells. The interaction between Sox2 and ß-catenin was substantially different between the two cell subsets separated based on their differential responsiveness to a Sox2 reporter. Specifically, while ß-catenin binds to Sox2 in the nuclear fraction of cells showing reporter-responsiveness (i.e. RR cells), this interaction was not detectable in those that were reporter-unresponsive (i.e. RU cells). In RR but not in RU cells, siRNA knockdown of ß-catenin significantly upregulated the Sox2 transcriptional activity, enhanced its DNA binding and increased the expression of its target genes. Correlating with these findings, while inhibition of ß-catenin significantly downregulated the mammosphere formation efficiency in RU cells, this treatment paradoxically increased that of RR cells. To conclude, we identified that ß-catenin is an important binding partner of Sox2 and a regulator of its transcriptional activity in a small subset of BC cells. The interaction between Sox2 and ß-catenin provides a novel mechanism underlying the functional dichotomy of BC cells, which carries potential therapeutic implications.

Sox2 suppresses the invasiveness of breast cancer cells via a mechanism that is dependent on Twist1 and the status of Sox2 transcription activity.

BACKGROUND: Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood. METHODS: In-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1. RESULTS: We found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in 'reporter un-responsive' cells (RU cells) but not 'reporter responsive' cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent. CONCLUSION: Sox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications.

Luteolin is a novel p90 ribosomal S6 kinase (RSK) inhibitor that suppresses Notch4 signaling by blocking the activation of Y-box binding protein-1 (YB-1).

Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44(+) cells. Luteolin's efficacy against drug-resistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched populations. ChIP-on-ChIP experiments in SUM149 cells revealed a 12-fold enrichment of YB-1 binding to the Notch4 promoter. We chose to pursue this because there are several reports indicating that Notch4 maintains cells in an undifferentiated, TIC state. Herein we report that silencing YB-1 with siRNA decreased Notch4 mRNA. Conversely, transient expression of Flag:YB-1(WT) or the constitutively active mutant Flag:YB-1(D102) increased Notch4 mRNA. The levels of Notch4 transcript and the abundance of the Notch4 intracellular domain (N4ICD) correlated with activation of P-RSK(S221/7) and P-YB-1(S102) in a panel of TNBC cell lines. Silencing YB-1 or RSK reduced Notch4 mRNA and this corresponded with loss of N4ICD. Likewise, the RSK inhibitors, luteolin and BI-D1870, suppressed P-YB-1(S102) and thereby reduced Notch4. In conclusion, inhibiting the RSK/YB-1 pathway with luteolin is a novel approach to blocking Notch4 signaling and as such provides a means of inhibiting TICs.

Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity.

Sox2 (sex-determining region Y-box protein 2) is a transcription factor regulating pluripotency in embryonic stem cells. Sox2 is aberrantly expressed in breast and other cancers, though its biological significance remains widely unexplored. To understand the significance of this aberrancy, we assessed the transcription activity of Sox2 in two Sox2-expressing breast cancer cell lines, MCF7 and ZR751, using a lentiviral Sox2 GFP reporter vector. Surprisingly, Sox2 transcription activity, as measured by GFP expression encoded in a Sox2 reporter construct, was detectable only in a small subset of cells in both cell lines. Purification of GFP+ cells (cells with Sox2 activity) and GFP- cells (cells without Sox2 activity) was enriched for two phenotypically distinct cell populations in both MCF7 and ZR751 cell lines. Specifically, GFP+ cells formed significantly more colonies in methylcellulose and more mammospheres in vitro compared to GFP- cells. These phenotypic differences are directly linked to Sox2 as siRNA knockdown of Sox2 in GFP+ cells abolished these abilities. To provide a mechanistic explanation to our observations, we performed gel shift and chromatin immunoprecipitation studies; Sox2 was found to bind to its DNA binding consensus sequence and the promoters of Cyclin D1 and Nanog (two known Sox2 downstream targets) only in GFP+ cells. GFP+ cells also up-regulated CD49f, phospho-GSK3ß, and ß-catenin. In summary, we have identified two novel phenotypically distinct cell subsets in two breast cancer cell lines based on their differential Sox2 transcription activity. We demonstrate that Sox2 transcription activity, and not its protein expression alone, underlies the tumorigenicity and cancer stem cell-like phenotypes in breast cancers.

Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates.

Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction.

Role of the intramolecular hydrogen bond network in the inhibitory power of chymotrypsin inhibitor 2.

A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues involved in intramolecular interactions that shape and constrain the binding loop, were studied to determine their relative importance for inhibition of the serine protease subtilisin BPN', and for resistance of the inhibitor to proteolysis. These functional properties were investigated in tandem with the crystal structures of the mutant inhibitor-enzyme complexes. A dense hydrogen bonding network that supports the binding loop in the vicinity of the scissile bond was found to be important both for enzyme affinity and for stability to proteolysis. Structural analysis, in combination with biochemical measurements, allows differentiation of the structural components most important for resistance to proteolysis and/or binding. The most critical participating residues in the network were found to be Thr-58, Glu-60, Arg-65, and Gly-83. Glu-60 is more important for resistance to proteolysis than for binding, while Arg-65 and two other Arg residues play a greater role in binding than in resistance to proteolysis. Structural comparisons reveal a wide variety of subtle conformational changes in response to mutation, with built-in robustness in the hydrogen bond network, such that loss of one contact is compensated by other new contacts.