Keratin 19 (K19) belongs to the keratin family of proteins, which maintains structural integrity of epithelia. In cancer, K19 is highly expressed in several types where it serves as a diagnostic marker. Despite the positive correlation between higher expression of K19 in tumor and worse patient survival, the role of K19 in breast cancer remains unclear. Therefore, we ablated K19 expression in MCF7 breast cancer cells and found that K19 was required for cell proliferation. Transcriptome analyses of KRT19 knockout cells identified defects in cell cycle progression and levels of target genes of E2F1, a key transcriptional factor for the transition into S phase. Furthermore, proper levels of cyclin dependent kinases (CDKs) and cyclins, including D-type cyclins critical for E2F1 activation, were dependent on K19 expression, and K19-cyclin D co-expression was observed in human breast cancer tissues. Importantly, K19 interacts with cyclin D3, and a loss of K19 resulted in decreased protein stability of cyclin D3 and sensitivity of cells towards CDK inhibitor-induced cell death. Overall, these findings reveal a novel function of K19 in the regulation of cell cycle program and suggest that K19 may be used to predict the efficacy of CDK inhibitors for treatments of breast cancer.

The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12+Gal3+T+CD24+ cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.

Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration.

Species generalization in the profound, modality-specific effects of Hedgehog pathway inhibition (HPI) in taste organ homeostasis and sensation is shown. With the HPI, cancer drug sonidegib, we demonstrate that the rat taste system, in addition to mouse, is regulated by Hedgehog signaling. After sonidegib treatment for 16-36 days in rat, there is loss of taste buds (TB) in soft palate, in fungiform (FP) and circumvallate papillae (CV), and elimination of taste responses from chorda tympani and glossopharyngeal nerves. The retained innervation in FP and CV during HPI cannot sustain TB. Responses to tactile stimuli are not altered, and temperature responses are reduced only after 28 days treatment, demonstrating modality-specific effects. Rat FP and neural effects are similar to those in mouse whereas TB and neural response effects from the rat CV are much more severe. When recovery is introduced in mouse after prolonged, 48 days HPI, the TB in CV are restored whereas those in FP are not. Overall, Hedgehog signaling regulation is shown to generalize to the rat taste system, and the modality-specific controls in taste organ sensation are affirmed. The reported, debilitating taste disturbances in patients who use HPI drugs can be better understood based on these data.

The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies.

Radiation therapy for head and neck cancers results in permanent damage to the saliva producing acinar compartment of the salivary gland. To date, a pure pro-acinar cell line to study underlying mechanisms of acinar cell differentiation in culture has not been described. Here, we report the establishment of a pro-acinar (mSG-PAC1) and ductal (mSG-DUC1) cell line, from the murine submandibular salivary gland (SMG), which recapitulate developmental milestones in differentiation. mSG-DUC1 cells express the ductal markers, keratin-7 and keratin-19, and form lumenized spheroids. mSG-PAC1 cells express the pro-acinar markers SOX10 and aquaporin-5. Using the mSG-PAC1 cell line, we demonstrate that FGF2 regulates specific steps during acinar cell maturation. FGF2 up-regulates aquaporin-5 and the expression of the α3 and α6 subunits of the α3β1 and α6β1 integrins that are known to promote SMG morphogenesis and differentiation. mSG-DUC1 and mSG-PAC1 cells were derived from genetically modified mice, homozygous for floxed alleles of the integrin α3 subunit. Similar to SMGs from α3-null mice, deletion of α3 alleles in mSG-PAC1 cells results in the up-regulation of E-cadherin and the down-regulation of CDC42. Our data indicate that mSG-DUC1 and mSG-PAC1 cells will serve as important tools to gain mechanistic insight into salivary gland morphogenesis and differentiation.

In some fibroblasts, casein kinase 1α (CK1α) is localized to nuclear speckles, which are sub-nuclear compartments supplying splicing factors, whereas it is recruited on keratin filaments in colorectal cancer cells such as DLD1 cells. In order to obtain a deeper understanding of why CK1α is localized to these different subcellular sites, we herein elucidated the mechanisms underlying its localization to nuclear speckles. CK1α and FAM83H were localized to nuclear speckles in RKO and WiDr colorectal cancer cells, which do not express simple epithelial keratins, and in DLD1 cells transfected with siRNAs for type I keratins. The localization of FAM83H to nuclear speckles was also detected in colorectal cancer cells with a poorly organized keratin cytoskeleton in colorectal cancer tissues. Using an interactome analysis of FAM83H, we identified SON, a protein present in nuclear speckles, as a scaffold protein to which FAM83H recruits CK1α. This result was supported by the knockdown of FAM83H or SON delocalizing CK1α from nuclear speckles. We also found that CK1δ and ε are localized to nuclear speckles in a FAM83H-dependent manner. These results suggest that CK1 is recruited to nuclear speckles by FAM83H and SON in the absence of an intact keratin cytoskeleton.

Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.