Hsi Hui Lin

Mechanosensing machinery for cells under low substratum rigidity.

Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates beta(1)-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered beta(1)-integrin (V737N), but not constitutively active beta(1)-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess beta(1)-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers beta(1)-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of beta(1)-integrin clustering enhances the interaction between beta(1)-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.

Mouse Kidney Progenitor Cells Accelerate Renal Regeneration and Prolong Survival after Ischemic Injury

Acute tubular necrosis is followed by regeneration of damaged renal tubular epithelial cells, and renal stem cells are supposed to contribute to this process. The purpose of our study is to test the hypothesis that renal stem cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal stem cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include (1) spindle‐shaped morphology, (2) self‐renewal of more than 100 passages without evidence of senescence, and (3) expression of Oct‐4, Pax‐2, Wnt‐4, WT‐1, vimentin, α‐smooth muscle actin, CD29, and S100A4 but no SSEA‐1, c‐kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intrarenal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone, and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult stem cell population, which may contribute to the renal repair and prolong survival after ischemic injury. STEM CELLS 2010;28:573–584

A tale of two collagen receptors, integrin β1 and discoidin domain receptor 1, in epithelial cell differentiation

As increase in collagen deposition is no longer taken as simply a consequence but, rather, an inducer of disease progression; therefore, the understanding of collagen signal transduction is fundamentally important. Cells contain at least two types of collagen receptors: integrins and discoidin domain receptors (DDRs). The integrin heterodimers α(1)β(1), α(2)β(1), α(10)β(1), and α(11)β(1) are recognized as the non-tyrosine kinase collagen receptors. DDR1 and 2, the tyrosine kinase receptors of collagen, are specifically expressed in epithelium and mesenchyme, respectively. While integrin β(1) and DDR1 are both required for cell adhesion on collagen, their roles in epithelial cell differentiation during development and disease progression seem to counteract each other, with integrin β(1) favoring epithelium mesenchyme transition (EMT) and DDR1 inducing epithelial cell differentiation. The in vitro evidence shows that the integrin β(1) and DDR1 exert opposing actions in regulation of membrane stability of E-cadherin, which itself is a critical regulator of epithelial cell differentiation. Here, we review the functional roles of integrin β(1) and DDR1 in regulation of epithelial cell differentiation during development and disease progression, and explore the underlining mechanisms regarding to the regulation of membrane stability of E-cadherin.

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young’s modulus (Eeff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.

Mechanosensitive store‐operated calcium entry regulates the formation of cell polarity

Ca2+‐mediated formation of cell polarity is essential for directional migration which plays an important role in physiological and pathological processes in organisms. To examine the critical role of store‐operated Ca2+ entry, which is the major form of extracellular Ca2+ influx in non‐excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca2+ was concentrated at the rear end of cells and that extracellular Ca2+ influx was important for cell polarization. Inhibition of store‐operated Ca2+ entry using specific inhibitors disrupted the formation of cell polarity in a dose‐dependent manner. Moreover, the channelosomal components caveolin‐1, TRPC1, and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin‐1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, store‐operated Ca2+ entry activity, and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel. J. Cell. Physiol. 230: 2086–2097, 2015.

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.

Matrix-Stiffness-Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis.

The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression.

Mechanical coupling of cytoskeletal elasticity and force generation is crucial for understanding the migrating nature of keloid fibroblasts

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra‐cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano‐signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration‐related disease such as keloid.

Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and β1 integrin

Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that β1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated β1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-β-cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and β1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phospho-mimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in β1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering β1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between β1 integrin and Cav1. Finally, co-expression of this auto-clustering β1 integrin and Cav1-Y14D synergistically enhanced cell spreading, and FA assembly in HEK293T cells cultured on either stiff ( > G Pa) or soft (0.2 kPa) matrices. Collectively, these results suggest that matrix stiffness governs the expression of β1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.

Activation of caspase‐8 and Erk‐1/2 in domes regulates cell death induced by confluence in MDCK cells

Under normal culture conditions, cells adhere to culture dish, spread out, proliferate, and finally cover all areas and reach confluence. During the confluent stage, cell proliferation ceases and differentiation is enhanced. Meanwhile, cell death also appears as the monolayer confluence proceeds. To delineate the mechanism of cell death induced by the confluent process, we employed Madin‐Darby canine kidney (MDCK) cells. When approaching confluence, MDCK cells exhibited increase the levels of caspase‐2 and enhanced the activity of caspase‐8. Using various caspase inhibitors to block apoptosis, we found that only z‐VAD‐fmk and z‐IETD‐fmk can inhibit confluent cell death, indicating that confluent cell death is mediated by activation of caspase‐8. Overexpression of Bcl‐2 inhibited confluent cell death, suggesting the involvement of mitochondria‐dependent pathway in confluent cell death. Interestingly, the activity of phospho‐Erk (p‐Erk) was initially decreased before confluence, but markedly increased after confluence. Immunofluorescence staining studies showed that p‐Erk was expressed exclusively on dome‐forming cells that underwent apoptosis. Treatment of confluent MDCK cells with PD98059 and UO126, the inhibitors of MEK, enhanced apoptosis as well as activity of caspase‐8. These data indicate that elevation of p‐Erk activity during confluence may serve to suppress confluent cell death. Taken together, activation of caspase‐8 contributes to and results in confluent cell death, whereas elevated p‐Erk activity serves to prevent confluent cell death by regulating activation of caspase‐8. J. Cell. Physiol. 211: 174–182, 2007.