Sonali Pal-Ghosh

Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse

Mice lacking syndecan-1 are viable, fertile and have morphologically normal skin, hair and ocular surface epithelia. While studying the response of these mice to corneal epithelial and skin wounding, we identified defects in epithelial cell proliferation and regulation of integrin expression. mRNA profiling of corneal epithelial tissues obtained from wild-type and syndecan-1-/- mice suggest that these defects result from differences in overall gene transcription. In the cornea, syndecan-1-/- epithelial cells migrate more slowly, show reduced localization of α9 integrin during closure of wounds and fail to increase their proliferation rate 24 hours after wounding. In the skin, we did not document a migration defect after full thickness wounds but did observe cell proliferation delays and reduced localization of α9 integrin in the syndecan-1-/- epidermis after dermabrasion. Despite increased cell proliferation rates in the uninjured syndecan-1-/- epidermis and the corneal epithelium, morphologically normal epithelial thickness is maintained prior to injury; however, wounding is accompanied by prolonged hypoplasia in both tissues. Analyses of integrin protein levels in extracts from full thickness skin, revealed increased levels of α3 and α9 integrins both prior to injury and after hair removal in syndecan-1-/- mice but no increase 2 days after dermabrasion. These data for the first time show involvement of α9 integrin in skin wound healing and demonstrate essential roles for syndecan-1 in mediating cell proliferation and regulation of integrin expression in normal and wounded epithelial tissues.

Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse.

Adult corneal epithelial stem cells (CESCs) have been shown to reside at the periphery of the cornea at a site called the corneoscleral junction or limbus. Although studies have shown that these cells are slow cycling, their molecular characteristics are not well understood. Using a whole‐mount procedure, we show that whereas α9‐integrin is present in a subset of the basal cells at the corneal limbus and absent in the central cornea, β1‐, β4‐, α3‐, and α6‐integrins are more highly expressed overall in central corneal basal cells. To characterize CESCs based on their slow‐cycling nature, we simultaneously evaluated 5‐bromo‐2‐deoxyuridine (BrdU) label‐retaining cells (LRCs) and integrin expression (α9, β1, and β4) in a total of 1,889 cells at the limbus of adult mice that had been injected as neonates with BrdU. Whereas the LRCs were usually observed adjacent to α9‐integrin‐positive cells, most LRCs were α9‐integrin–negative and expressed high levels of β1‐ and β4‐integrin. In addition, we observed more BrdU‐positive LRCs at the superior and inferior quadrants of adult mouse corneas than at the nasal and temporal quadrants, and determined that 0.94 to 3.6% of the limbal basal cells were slow cycling. We conclude from these data that the slow‐cycling LRCs in the adult mouse cornea are enriched in cells that express high levels of β1‐ and β4‐integrin and little α9‐integrin.

Loss of Integrin α9β1 Results in Defects in Proliferation, Causing Poor Re-Epithelialization during Cutaneous Wound Healing

The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and beta1 integrins are important in this process. We previously reported a marked increase in integrin alpha9beta1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte alpha9beta1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin alpha9beta1 knockout mice. We now report that in conditional integrin alpha9 knockout (K14-alpha9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin alpha9beta1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-alpha9 null mice compared with alpha9(fl/-) mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-alpha9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that alpha9beta1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.

Reduced migration, altered matrix and enhanced TGFbeta1 signaling are signatures of mouse keratinocytes lacking Sdc1.

We have reported previously that syndecan-1 (Sdc1)-null mice show delayed re-epithelialization after skin and corneal wounding. Here, we show that primary keratinocytes obtained from Sdc1-null mice and grown for 3-5 days in culture are more proliferative, more adherent and migrate more slowly than wt keratinocytes. However, the migration rates of Sdc1-null keratinocytes can be restored to wild-type levels by replating Sdc1-null keratinocytes onto tissue culture plates coated with fibronectin and collagen I, laminin (LN)-332 or onto the matrices produced by wild-type cells. Migration rates can also be restored by treating Sdc1-null keratinocytes with antibodies that block α6 or αv integrin function, or with TGFβ1. Antagonizing either β1 integrin function using a function-blocking antibody or TGFβ1 using a neutralizing antibody reduced wild-type keratinocyte migration more than Sdc1-null keratinocyte migration. Cultures of Sdc1-null keratinocytes accumulated less collagen than wild-type cultures but their matrices contained the same amount of LN-332. The Sdc1-null keratinocytes expressed similar total amounts of eight different integrin subunits but showed increased surface expression of αvβ6, αvβ8, and α6β4 integrins compared with wild-type keratinocytes. Whereas wild-type keratinocytes increased their surface expression of α2β1, αvβ6, αvβ8, and α6β4 after treatment with TGFβ1, Sdc1-null keratinocytes did not. Additional data from a dual-reporter assay and quantification of phosphorylated Smad2 show that TGFβ1 signaling is constitutively elevated in Sdc1-null keratinocytes. Thus, our results identify TGFβ1 signaling and Sdc1 expression as important factors regulating integrin surface expression, activity and migration in keratinocyte and provide new insight into the functions regulated by Sdc1.

MMP9 cleavage of the β4 integrin ectodomain leads to recurrent epithelial erosions in mice

Integrin α6β4 is an integral membrane protein within hemidesmosomes and it mediates adhesion of epithelial cells to their underlying basement membrane. During wound healing, disassembly of hemidesmosomes must occur for sheet movement-mediated cell migration. The mechanisms of disassembly and reassembly of hemidesmosomes are not fully understood. The current study was initiated to understand the underlying cause of recurrent corneal erosions in the mouse. Here, we show that in vivo: (1) MMP9 levels are elevated and β4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the β4 ectodomain is missing from sites where erosions develop; and (3) β4 cleavage can be reduced by inhibiting MMP activity. Although β4, α3 and β1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that β4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the β4 integrin extracellular domain, generating a 100 kDa β4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of β4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6β4 and β1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that β4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.

BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds.

Genetically engineered mice are usually produced on a mixed genetic background and can be derived from several mouse strains including 129SvJ, C57BL6, and BALB/c. To determine whether differences in recurrent corneal epithelial erosions (RCEEs), corneal epithelial stem cell deficiency (CESCD), and cell migration rate vary between two different mouse strains (BALB/c and C57BL6), 8-week mice were subjected to 1.5 (small) or 2.8mm (large) manual debridement wounds and allowed to heal for 4 weeks. Syndecan-1 (sdc-1) null mice backcrossed seven generations onto a BALB/c genetic background were also included in the RCEE and CESCD studies to permit comparisons between genotypes within a single strain. After sacrifice, corneas were assessed for the presence of recurrent erosions; no fewer than 15 corneas were used for each strain or genotype studied. Data show that the frequency of recurrent erosions after small wounds was 81+/-9% in the C57BL6 mice, 73+/-2% in the BALB/c mice, and 32+/-6% in sdc-1 null mice. Neither strain developed CESCD after small wounds. The frequency of erosions after large wounds was greater (88+/-8%) in the C57BL6 mice compared to BALB/c (60+/-2%), and sdc-1 null mice (32+/-5%). Four weeks after the large wounds, fixed, flat mounted corneas were assessed for evidence of CESCD with antibodies against the conjunctival keratin K8 and the goblet cell marker, the mucin Muc5AC. The frequency of CESCD 4 weeks after the large wounds was significantly greater in the C57BL6 mice than in the BALB/c or sdc-1 null mice. To assess cell migration rates, corneas were subjected to 1.5mm wounds and allowed to heal for 12, 15, 18, 21, and 24h. After sacrifice, corneas were stained with Richardson stain (BALB/c) or propidium iodide (C57BL6) to assess reepithelialization rates. While reepithelialization rates were similar for the early times after wounding, by 24h the C57BL6 corneas had healed faster: 16 of 30 corneas from the C57BL6 mice were closed compared to 9 of 30 of the BALB/c wounds. BALB/c corneas appeared larger overall compared to C57BL6 corneas; measurements of the overall mass of the enucleated eyes and diameters of the flat-mounted corneas confirmed that C57BL6 eyes and corneas were 6.8% and 4.4% smaller respectively than those of BALB/c mice even though the masses of the two mouse strains at 8 weeks of age were identical. Using BrdU to label dividing cells, we found that 18 h after wounding, C57BL6 and BALB/c corneal epithelia showed similar numbers of proliferating cells. To determine if the enhanced corneal epithelial cell migration rate seen in the C57BL6 mice was specific to the cornea, we conducted time-lapse studies to assess random cell migration rates in vitro using primary cultures of mouse epidermal keratinocytes. Consistent with the in vivo data, epidermal keratinocytes derived from BALB/c mice migrated 60% slower than C57BL6 cells. These data prove that strain-specific differences in cell migration rate in vivo are present in the cornea and are accompanied by differences in the frequencies of recurrent erosions and corneal epithelial stem cell deficiency.

Corneal Goblet Cells and Their Niche: Implications for Corneal Stem Cell Deficiency

Goblet cells are terminally differentiated cells secreting mucins and antibacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow‐cycling corneal progenitor cells, proliferating cells, nonproliferating cells, and postmitotic differentiated K12+Muc5ac+ goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency. Stem Cells2012;30:2032–2043

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.

Primary dermal fibroblasts derived from sdc-1 deficient mice migrate faster and have altered alphav integrin function.

The goal of this study is to determine whether dermal fibroblasts lacking syndecan‐1 (sdc1) show differences in integrin expression and function that could contribute to the delayed skin and corneal wound healing phenotypes seen in sdc‐1 null mice. Using primary dermal fibroblasts, we show that after 3 days in culture no differences in α‐smooth muscle actin were detected but sdc‐1 null cells expressed significantly more αv and β1 integrin than wildtype (wt) cells. Transforming growth factor β1 (TGFβ1) treatment at day 3 increased αv‐ and β1‐integrin expression in sdc‐1 null cells at day 5 whereas wt cells showed increased expression only of αv‐integrin. Using time‐lapse studies, we showed that the sdc‐1 null fibroblasts migrate faster than wt fibroblasts, treatment with TGFβ1 increased these migration differences, and treatment with a TGFβ1 antagonist caused sdc‐1 null fibroblasts to slow down and migrate at the same rate as untreated wt cells. Cell spreading studies on replated fibroblasts showed altered cell spreading and focal adhesion formation on vitronectin and fibronectin‐coated surfaces. Additional time lapse studies with β1‐ and αv‐integrin antibody antagonists, showed that wt fibroblasts expressing sdc‐1 had activated integrins on their surface that impeded their migration whereas the null cells expressed αv‐containing integrins which were less adhesive and enhanced cell migration. Surface expression studies showed increased surface expression of α2β1 and α3β1 on the sdc‐1 null fibroblasts compared with wt fibroblasts but no significant differences in surface expression of α5β1, αvβ3, or αvβ5. Taken together, our data indicates that sdc‐1 functions in the activation of αv‐containing integrins and support the hypothesis that impaired wound healing phenotypes seen in sdc‐1 null mice could be due to integrin‐mediated defects in fibroblast migration after injury.

Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis

Syndecan‐1 (sdc‐1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc‐1 delays wound healing, reduces migration, and increases Transforming growth factor β (TGFβ) 1 expression. In this study we show that sdc‐1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc‐1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc‐1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc‐1 null mice expressed suprabasal α3 and β4 integrins; suprabasal β4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol‐12‐myristate‐13‐acetate (TPA) did not differ among the genotypes, sdc‐1 null mice had an enhanced inflammatory response and retained higher levels of total TGFβ1 within their skin after TPA treatment. sdc‐1 null keratinocytes, transduced in vitro by oncogenic rasHa, expressed higher levels of β4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt rasHa‐transduced keratinocytes. When rasHa‐transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc‐1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc‐1 could be a useful marker for progression in neoplastic skin lesions.